LABORATORY  EXERCISES 
IN  BACTERIOLOGY 


SMITH 


LESSONS  AND  LABORATORY  EXERCISES 
IN  BACTERIOLOGY. 


SMITH. 


LESSONS   AND    LABORATORY 
EXERCISES 


IN 


BACTERIOLOGY 

AN  OUTLINE  OF  TECHNICAL  METHODS  INTRODUC- 
TORY TO  THE  SYSTEMATIC  STUDY  AND 
IDENTIFICATION  OF  BACTERIA, 

ARRANGED  FOR  THE  USE  OF  STUDENTS 


ALLEN   J.   SMITH,  M.D., 

h 

PROFESSOR    OF    PATHOLOGY    IN   THE    UNIVERSITY    OF    TEXAS,  GALVESTON. 


PHILADELPHIA 

P.    BLAKISTON'S   SON   &  CO 

1012   WALNUT  STREET 
1902 


THt  LibRARY  OF 
CONGRESS, 

T/.-o  Copies  Received 

NOV.    8   1902 

Copyright    £ntry 

J 

LASS  CA_-  xXc.  No 


COPY  A. 


COPYRIGHT,  1902,  BY  ALLEN  J.  SMITH. 


PRESS  OF-WM.   F.   FELL  &  CO.. 

I22O-24   SANSOM   ST.. 

PHILADELPHIA. 


PREFACE. 


The  following  pages  were  originally  arranged  as  a  series  of  exercises  to 
be  carried  out  by  the  class  in  the  University  of  Texas  under  the  guidance  of 
an  assistant,  the  hours  of  work  of  the  several  classes  so  overlapping  that  it 
was  impossible  for  the  writer  to  give  his  personal  attention  to  the  class  in 
this  laboratory,  for  the  work  in  which  he  was,  however,  responsible.  The 
exercises  were  outlined  daily  upon  the  blackboard,  and  verbal  instructions  as 
to  their  purpose  and  the  manner  of  procedure  given,  as  required,  to  the  class. 
The  arrangement  being  found  well  adapted  to  systematic  work,  it  has  been 
continued,  with  changes  from  and  some  additions  to  the  original  scheme, 
for  several  years ;  and  recently,  at  the  request  of  some  of  his  students  and 
with  the  thought  that  perhaps  others  might  find  some  such  definite  ar- 
rangement of  work  of  use  in  teaching,  the  writer  has  written  out  these  exer- 
cises for  publication.  It  has  seemed  advantageous  that  in  their  published 
form  there  should  accompany  the  v^ractieal,  exercises  such  explanatory 
matter  as  would  adapt  the  book  as  a  student's  laboratory  outline  guide. 
It  would  be  beyond  the  writer's  intention  to  make  the  work  a  compendium 
of  methods,  and  the  instructions,  although  forming  a  considerable  bulk  of 
the  volume,  include  only  such  as  from  his  experience  he  has  adopted  as  best 
suited  in  class  work. 

There  are  arranged  blank  pages  upon  which  notes  of  the  outcome  of  the 
various  experiments  and  record  of  special  instruction  as  to  technique  may 
be  added ;  and  as  an  appendix  a  blank  form  is  printed,  following  which  as  a 
form  at  the  close  of  the  work  should  be  recorded  the  data  ascertained  in 
connection  with  the  more  important  forms  of  microorganisms  which  have 
been  studied  in  the  exercises.*  It  has  been  the  writer's  custom  to  have 
each  student  carry  along  with  the  regular  class  work  during  the  last  two  or 
three  weeks  some  simple  independent  task,  as  the  bacterial  analysis  of  a 
water-supply,  of  milk,  soil,  or  air,  in  which  an  enumeration  of  the  bacteria 
found  in  a  definite  quantity  of  the  substance  examined  is  required,  together 
with  the  identification  of  one  or  more  forms  of  the  organisms  encountered, 
and  such  study  of  their  pathogenic  influence  as  time  permits.  The  records 
of  such  work  also  may  well  be  made  after  the  blank  form  in  the  appendix. 

*  Copies  of  this  form  maybe  obtained  from  P.  Blakiston's  Son  &  Co.,  Philadelphia. 

iii 

268459 


iv  PREFACE. 

More  than  any  other  one  measure,  this  practice  has  seemed  of  value  in 
giving  the  student  confidence  in  his  powers  of  observation  and  reliance 
on  his  application  of  methods. 

It  has  come  to  be  a  question  as  to  how  much  of  the  general  subject  shall 
be  taught  in  the  brief  course  permissible  in  the  crowded  medical  curriculum. 
In  the  earlier  days  of  his  teaching  the  writer  paid  more  attention  to  the 
detailed  characteristics  of  the  important  bacteria,  especially  the  pathogens, 
than  to  technical  work;  this  more  because  of  the  lack  of  a  satisfactory 
system  of  classification  of  the  bacteria  and  a  chaotic  condition  of  the  gen- 
eral study  than  because  he  failed  to  appreciate  the  need  of  such  system. 
In  the  more  recent  years,  however,  much  advance  has  been  made  in  these 
lines,  and  it  is  more  in  harmony  with  the  purpose  of  education  that  the 
student  should  be  taught  in  the  laboratory  the  methods  of  demonstration 
of  bacterial  characters  and  properties,  and  a  proper  manner  of  investiga- 
tion and  observation,  than  that  he  should  devote  his  time  and  attention 
to  isolated  facts  which  in  many  instances  are  better  brought  to  his  com- 
prehension in  connection  with  his  work  in  medicine  or  surgery.  The  writer 
has  endeavored  in  selection  of  the  illustrative  work  to  embody  the  more 
important  points  which  clinical  study  demands,  but  in  no  other  way 
than  as  important  illustrations.  Such  special  points  cannot,  of  course, 
be  too  often  brought  forward  or  too  much  impressed,  but  they  will  prob- 
ably be  best  remembered  from  their  relations  to  clinical  instruction. 

There  are  a  number  of  works  of  a  systematic  character  adapted  to 
bacterial  determination  and  identification,  although  the  perfect  system  of 
bacteriologic  classification  is  probably  far  in  the  future;  during  the  past 
year  the  writer  has  become  familiar  with  the  many  excellent  features  of 
Chester's  Determinative  Bacteriology,  and  in  the  arrangement  of  the  follow- 
ing pages  the  latter  was  in  mind  as  a  suitable  reference  book  for  the  student 
in  working  out  the  identification  of  unknown  species  and  for  inquiry  as  to 
the  important  cultural  characteristics  of  known  bacteria.  The  admirable 
work  of  Neumann  and  Lehmann  in  its  American  edition  is  equally  to  be 
commended,  particularly  in  its  technical  and  descriptive  portions,  for  class 
purposes. 

As  outlined,  the  entire  series  of  exercises  embodied  in  the  following 
pages  may  be  carried  out  in  eight  or  nine  weeks,  each  student  working  at 
least  ten  hours  each  week.  It  will  be  necessary,  however,  that  the  in- 
structor inaugurate  such  work  as  will  require  time  for  its  completion  before 
the  completion  of  preceding  exercises;  and  it  is  well  to  have  the  student 
understand  that  the  order  of  work  corresponds  rather  with  the  end  than 
with  the  beginning  of  each  task. 

There  is,  of  course,  nothing  original  in  the  book  either  in  the  matter  or 


PREFACE  v 

in  the  mode  of  presentation,  both  of  which  are  more  fully  and  more  satis- 
factorily covered  in  the  systematic  texts  upon  the  subject.  Especial 
acknowledgment  should  be  made  for  the  use  of  the  series  of  descriptive 
terms  and  the  accompanying  illustrations  on  pages  172-184,  which  have 
been  taken  from  Chester's  Determinative  Bacteriology,  1901,  with  the  kind 
permission  of  its  author.  It  is  only  as  an  attempt  toward  fixation  of  sys- 
tematic procedures  in  class  work  that  the  writer  can  hope  to  have  added  in 
the  least  to  the  advance  of  a  subject  in  which  so  much  good  work  has  been 
done  by  a  host  of  laborers.  Should  this  prove  true  it  will  not  be  a  source 
of  regret  that  this  scheme,  imperfect  and  incomplete  as  it  is,  has  thus  been 
made  public.  A.  J.  S. 

GALVESTON,  TEXAS,  August  79,  1902. 


TABLE  OF  CONTENTS. 


LESSON  I.  PACK. 

INTRODUCTORY,  9-14 

Plan  of  Work,  9;  Laboratory  Cleanliness,  10;  Laboratory  Provisions,  10 
Place  of  Bacteria  in  Nature,  12;  Preliminary  Classification,  12;  Impor- 
tance to  Man,  14. 

LESSON   II. 

STERILIZATION, 16-56 

Occurrence  of  Bacteria  in  Nature,  16.  Sterilization:  Flaming,  18;  Dry- 
air  Oven,  20 ;  Boiling,  24 ;  Steam  Sterilization  by  Ordinary  Method,  by 
Autoclave,  and  by  Pasteurization,  24 ;  Determination  of  Thermal  Death- 
point,  32;  Sterilization  by  Filtration,  36;  Sterilization  by  Chemical 
Solutions,  40 ;  Determination  of  Disinfectant  and  Antiseptic  Values,  46 ; 
Disinfectant  Gases,  54. 

LESSON  III. 

PREPARATION  OF  TUBES,  FLASKS,  DISHES,  ETC.,  FOR  CULTURE  MEDIA, 58-68 

Tubes,  58;  Cleansing  Tubes,  58;  Plugging  Tubes,  60;  Sterilization  of 
Tubes,  62;  Special  Forms,  62;  Flasks:  Uses,  Forms,  Cleansing,  etc.,  64. 
Dishes :  Culture  Dishes,  Their  Preparation  and  Uses,  64 ;  Petri  Dishes,  66 ; 
Plates,  68. 

LESSON  IV. 

CULTURE  MEDIA,  70-106 

Purposes  and  Character,  70;  Reaction,  70.  Carbohydrate  Media:  Prepara- 
tion of  Potatoes  for  Dish  Culture,  72 ;  for  Tube  Culture,  76 ;  Glycerine 
Potato,  78;  Eisner's  Medium,  78;  Holz's  Potato  Gelatine,  78;  Other 
Carbohydrate  Media,  78.  Proteid  Media:  Bouillon,  80;  Sugar-contain- 
ing Bouillon,  82;  Distribution  of  Bouillon  and  Other  Liquid  Media  to 
Tubes,  etc.,  84;  Gelatine,  86;  Agar,  90;  Gelatine-agar,  92;  Glycerine 
Agar,  92 ;  Lactose-litmus-agar,  94 ;  Peptone  Solution,  94 ;  Rosolic  Acid- 
peptone  Solution,  94;  Blood-serum,  94;  Loeffler's  Medium,  104;  Milk, 
104;  Litmus  Milk,  106.  Preservation  of  Media,  106. 

LESSON    V. 

INOCULATION  OF  MEDIA  AND  CULTIVATION  OF  BACTERIA, 108-170 

Appliances  for  Inoculating:  Platinum  Needle,  108;  Swabs,  112;  Pipettes, 
114;  Syringes,  116;  Sternberg's  Bulbs,  116;  Blades,  118;  Forceps,  118; 
Particles,  118;  Fractional  Inoculations,  120.  Source  and  Collection  of 
Material  for  Inoculation,  122.  Cultures:  Tube  Cultures,  124;  Plate  Cul- 
tures, 124;  Dish  Cultures,  126;  Esmarch  Tubes,  128.  Illustrative  Proce- 
dures: Examination  of  Air,  128;  Examination  of  Water,  134;  Examina- 
tion of  Milk,  142;  Examination  of  Soil,  144;  Examination  of  Material 
from  Autopsy,  •  144.  Cultivation  of  Inoculated  Material:  Conditions  of 
Bacterial  Growth,  Selection  of  Nutrient,  148;  Moisture,  148;  Tempera- 
ture, 150;  Incubator,  150;  Thermostat,  152;  Gas-pressure  Regulator, 
156;  Atmosphere,  156;  Anaerobic  Jar,  160;  Anaerobic  Tubes,  160. 
Numerical  Estimation  of  Bacteria,  162. 


viii  TABLE   OF   CONTENTS. 

LESSON  VI.  PAGE. 

GROSS  APPEARANCE  OF  BACTERIAL  CULTURES, 172-184 

Colonies  of  Bacteria,  172;  Observed  Features,  172;  List  of  Cultural 
Characters,  174. 

LESSON  VII. 

INDIVIDUAL  BACTERIA,  THEIR  PHYSICAL  AND  CHEMICAL  CHARACTERISTICS,  . . .  186-244 
Microscope,  186;  Slides  and  Cover-glasses,  188;  Microscopic  Examina- 
tion of  Colonies,  188;  Hanging-drop  Preparations,  188;  Film  Prepara- 
tions, 192;  Staining  Reagents  and  Mixtures,  194;  Staining  Methods,  202. 
Physical  Characteristics  of  Bacteria:  Shape,  208 ;  Grouping,  210 ;  Structure, 
Capsules,  Flagella,  Nuclear  Granules,  212;  Size,  216;  Motility,  218;  Re- 
production, 220;  Formation  and  Germination  of  Spores,  222;  Determi- 
nation of  Spores,  224.  Chemical  Activities  of  Bacteria:  Pigment  Forma- 
tion, 226 ;  Photogenic  Power,  226 ;  Ferments :  Proteolytic  Ferments,  228 ; 
Diastasic  Ferments,  230;  Invertin  Ferments,  230;  Rennet  Ferments, 
230 ;  Gas  Production,  232 ;  Acid  Production,  234 ;  Alkaline  Production, 
234;  Alkaloidal  Products,  236;  Toxalbumins,  236;  Antitoxins,  236; 
Agglutination  Phenomenon,  238;  Indol  and  Phenol  Production,  240; 
Nitrifying  and  Denitrifying  Bacteria,  242. 

LESSON   VIII. 

ISOLATION  OF  BACTERIA  IN  PURE  CULTURE, 246-260 

Mechanical  Methods:  Plates,  246;  Petri  Dishes,  246;  Esmarch  Tubes, 
246;  Salomonsen's  Capillary  Tubes,  248:  Klebs'  Fractional  Method,  252. 
Physiologic  Methods:  Selection  by  Living  Animal  Tissues,  254;  Selection 
by  Culture  Media  and  Modifications,  256 ;  Selection  by  Culture  Tempera- 
ture, 258;  Selection  by  Culture  Atmosphere,  258;  Cohn's  Heating 
Method,  260. 

LESSON    IX. 

CLASSIFICATION  AND  IDENTIFICATION  OF  BACTERIA, 262-274 

Classification,  262 ;  Table  of  Classification  into  Genera,  264 ;  Analytic 
Table,  268 ;  List  of  Equivalent  Nomenclature  of  Genera,  272. 

LESSON    X. 

PATHOGENIC  ACTION  OF  BACTERIA,  276-290 

Importance  of  Study,  276 ;  Selection  of  Experiment  Animals,  278  ;  Hypo- 
dermic Inoculation  of  Animals,  278;  Intravenous  Inoculation,  280;  In- 
traperitoneal  Inoculation,  282;  Other  Forms  of  Inoculation,  282;  Care 
and  Observation  of  Experiment  Animals  after  Inoculation,  284 ;  Autopsy, 
286;  Immunity,  288. 


BACTERIOLOGIC  CHART. 

INDEX,  .  291 


LIST  OF  ILLUSTRATIONS. 


FIG.  PAGE. 

1 .  Form  Types  of  Bacteria 12 

2.  Position  in  which  Inoculating  Needle  is  Held  while  Cooling, 20 

3.  Proper  Position  of  Holding  Knife  after  Flaming, 20 

4.  Sectional  View  of  Hot-air  Oven, 22 

5.  Koch  $team  Sterilizer. — (From  —              — ), 26 

6.  Diagram  of  Arnold  Steam  Sterilizer. — (From  —             — ),    28 

7.  Sectional  View  of  Autoclave, 30 

8.  Glass  Bulb  to  Illustrate  Value  of  Cotton  Stoppers  as  Protection  from  Air 

Contamination,    36 

9.  Kitasato  Filter.— (From  -              — ) 38 

10.  Filter  Reversed  in  a  Percolator  Filled  with  Water  in  Order  to  Wash  Back 

Solid  Particles  Lodged  in  its  Wall, ,38 

11.  Test-tube   Brush, 58 

12.  Wire   Basket  for  Test-tubes.— (From   Williams) 60 

13.  Roux's  Potato  Tube, 60 

14.  Fermentation  Tube, 60 

15.  Fermentation  Tube, 60 

16.  Types  of  Culture  Flasks, 62 

1 7.  Types  of  Distribution  Flasks, 62 

18.  Culture   Dish. — (From  -              — ), -. 64 

19.  Petri   Dish.— (From   -               — ), 64 

20.  Metal  Box  for  Holding  Glass  Plates  During  Sterilization  in  Oven, 66 

21.  Set  of  Culture  Plates  and  Platforms, 66 

22.  Section  of  Potato  Intended  for  Dish  Culture, 74 

23.  Culture  Tube  Containing  Cylinder  of  Potato  Resting  on  Small  Glass  Rod, ....  76 

24.  Distribution  of  Liquid  Medium  from  Covered  Funnel  to  Culture  Tube, ....  86 

25.  Siphonage  of   Serum,    ! 86 

26.  Distribution  to  Culture  Tube  of  Liquid  Medium  from  Flask  A,  by  Pressure 

from  Siphon  Flask  B, -. 88 

27.  Hot-water  Filtration  Bucket, 92 

28.  Series  of  Tubes  Arranged  for  Collection  and  Sedimentation  of  Blood-serum,  96 

29.  Blood  Tube  with  Siphon  Arranged  to  Transfer  Serum  from  Jar  to  Sedimenta- 

tion Tubes;  Tube  a  to  be  Kept  above  Level  of  Serum  in  Last  Tube, 98 

30.  Distribution  from  Sedimentation  Tube  to  Culture  Tubes;  Tubule  a  Raised 

above  Red  Sediment  in  Sedimentation  Tube, 100 

31 .  Blood-serum  Inspissator,   Koch  Pattern, 102 

32.  Straight  Platinum  Wire  and  Platinum  Wire  Loop. — (From  —             — ),.  .  .  .  108 

33.  Proper  Mode  of  Holding  Tubes  of  Solid  Medium  in  Inoculation, 110 

34.  A.  Stroke  Culture.     B.  Puncture  Culture, '. 112 

35.  Sterile  Cotton  Swab  in  Tube,  with  Rubber  Cap  over  Mouth  of  Latter  for 

Greater  Protection  of  Swab  and  to  Prevent  Drying, 112 

ix 


LIST   OF   ILLUSTRATIONS. 


PAGE. 


36.  Laboratory  Water-bath  for  Melting  Solid  Media  in  Tubes, 114 

37.  Koch's  Inoculation  Syringe, 116 

38.  Sternberg  Bulb.— (From  -              — ), 118 

39.  Levelling  Tripod  Bearing  a  Dish  of  Cracked  Ice,  Covered  by  a  Glass  Plate. 

On  the  Plate  a  Level  and  Bell  Jar;  Beneath  the  Latter  a  Culture  Plate,.  .  126 

40.  A.  Sedgwick -Tucker  aero-bioscope.     B.  Glass  Tube  of  Hesse's  Apparatus,.  .  130 

41.  Collection  Filter,  for  Use  in  Concentrating  Bacteria  from  a  Large  Amount 

of  Water, 138 

42.  Bottle  in  Frame,  Arranged  for  Collecting  Water  from  Definite  Depths  and 

Replacing  Stopper  after  Entrance  of  Water, 140 

43.  Harpoon  for  Collecting  Soil  from  below  the  Surface  of  the  Ground, 144 

44.  Sectional  View  of  Incubator, 152 

45.  Thermostat, 154 

46.  Murrell  Gas  Pressure  Regulator, 156 

47.  Large    Class    Incubator    with   Interior  Warm- water    Tank,   and  Wire-cage 

Drawers,  Wall  of  Wood,  Covered  with  Felt, 158 

48.  Anaerobic  Jar, 160 

49.  Kip  Gas  Generator  and  Wash-bottle, 162 

50.  Mode  of  Counting  Colonies  in  an  Esmarch  Tube, 164 

51.  Gross  Cultural  Appearances,  Non-liquefying. — (After  Chester), 176 

52.  Types  of  Liquefying  Cultures. — (After  Chester), 178 

53.  Shapes  of  Colonies. — (After  Chester), 180 

54.  Shapes  of  Colonies. — (After  Chester), ; 180 

55.  Surface  Characters  of  Colonies. — (After  Chester), 182 

56.  Characters  of  Edges  of  Colonies. — (After  Chester), 184 

57.  Diagram  of  the  Hanging  Drop. — (From  -              —),  .  .  .  . 190 

58.  Stewart  Forceps  for  Cover-glasses. — (From  —             — ), 192 

59.  Tray  of  Staining  Tubes, 198 

60.  Grouping  of  Bacteria, ' 210 

61.  Structure  of  a  Bacterium. — (After  Migula), 212 

62.  Types  of  Capsule  Bacteria, 214 

63.  Flagellation  of   Bacteria, 214 

64.  Reproduction  of  Bacteria, 220 

65.  Types  of  Sporulation, ' 222 

66.  Types  of  Germination  of  Spores, 222 

67.  Salomonsen's  Capillary-Tubes  Inclosed  in  Protective  Glass  Tube,  with  Latter 

Attached  to  Card  on  which  are  Preserved  Notes  of  Colonies  Within, 250 

68.  Different  Forms  of  Animal  Holders, .  .  .280 


LESSONS  AND  LABORATORY  EXERCISES 
IN  BACTERIOLOGY. 


LESSON  I. 

INTRODUCTION. 

Plan  of  Work. — The  student  should  understand  that  the  following  pages  are  in- 
tended to  be  supplementary  to  the  general  lecture  instruction  in  bacteriology,  and  do 
not  in  any  sense  replace  such  instruction.  Nor  is  the  work  comparable  to  a  text-book 
upon  the  subject,  being  entirely  too  schematic  and  incomplete  for  use  as  such.  It  is 
intended  rather  that  the  instruction  outlined  in  the  following  pages  shall  serve  as  a  guide 
for  those  personal  exercises  and  experiments  which  shall  establish  the  verity  of  the  major 
propositions  of  the  systematic  teachings ;  and  shall  also  afford  some  scheme  in  outline 
for  personal  investigation  into  such  problems  in  the  subject  as  may  be  undertaken  by 
the  student  in  his  laboratory  experience.  The  pages  are  interleaved  with  blank  sheets  in 
order  that  upon  the  latter  may  be  noted  the  results  of  each  experiment ;  and  in  addition 
it  is  advised  that  these  blanks  be  employed  for  the  preservation  of  notes  of  all  detailed 
instruction  not  included  in  the  printed  page,  at  least  as  far  as  technique  is  concerned. 
For  the  general  discussions  of  bacteriology,  the  relations  of  the  schizomycetes  to  allied 
forms  of  vegetation,  their  relations  to  disease  phenomena,  and  the  many  and  varied 
problems  which  arise  in  the  subject  generally,  little  or  nothing  may  be  included  in  a 
book  of  the  scope  and  limitations  set  upon  this,  and  reference  in  such  lines  should  at  once 
be  made  to  the  systematic  texts  upon  the  subject. 

It  is  the  purpose  of  the  following  lessons  to  conduct  the  student  in  some  regular 
progression  through  those  processes  and  modes  of  operation  by  which  bacteria  may  be 
studied  for  the  recognition  of  the  individual  organism  and  its  classification  in  the  group 
of  the  cleft  fungi.  The  systematic  and  complete  study  of  this  or  that  form  is  not  sought, 
save  in  a  minor  degree,  since  there  must  in  the  present  crowded  condition  of  the  medical 
curriculum  be  little  possibility  to  hope  for  more  than  a  second-hand  knowledge  at  the 
hands  of  the  student  of  the  great  bulk  of  facts  accumulated  by  bacteriologists  in  regard 
to  individual  forms  of  bacteria,  whether  pathogenic  or  non-pathogenic ;  but  it  is  hoped 
that  the  student  who  has  followed  with  punctual  and  earnest  labor  the  processes  herein 
outlined  will  be  able,  by  some  such  analytic  key  as  is  presented,  to  undertake  with  intel- 
ligence and  expectation  of  success  the  determination  of  such  bacteriologic  growths  as  he 
may  encounter  in  his  later  and  fuller  studies.  In  other  words,  one  who  has  pursued 
faithfully  such  work,  although  he  may  not  be  regarded  as  a  bacteriologist,  should  be  in 
position,  by  practice  and  further  study  along  the  same  and  allied  lines,  to  become  com- 
petent as  such.  As  a  matter  of  practice  it  is  intended  that  at  least  a  few  of  the  more 
important  pathogenic  bacteria  and  such  unknown  organisms  as  may  be  met  in  material 
suggested  for  investigation  by  the  instructor  should  be  carried  through  the  outlined 
2  9 


10  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

methods,  and  at  the  close  of  the  book  a  tabulated  blank  is  inserted,  following  which  the 
preservation  of  the  notes  of  observation  in  such  work  should  be  made. 

Laboratory  Cleanliness. — In  the  pursuit  of  such  individual  practice  it  is  scarcely 
necessary  to  insist  upon  the  utmost  care  and  cleanliness  on  the  part  of  each  worker  at  all 
stages  of  the  work.  All  appliances  are  to  be  kept  in  constant  order  and  cleanliness,  and 
whenever  exposed  to  contamination  in  any  manner  should  be  subjected  to  thorough  ster- 
ilization in  the  most  suitable  manner.  Not  only  this,  but  the  individual  who  is  person- 
ally scrupulously  clean  is  not  only  the  most  successful  worker,  but  more  certainly  than 
the  careless  escapes  the  possible  danger  of  infection.  Suitable  receptacles  are  provided 
for  containing  discarded  material  in  disinfectant  solutions,  and  such  substances  are 
later  burned ;  and  each  student  should  bear  in  mind  as  a  constant  duty  to  see  that  all 
such  matter  is  promptly  and  properly  rendered  harmless.  Personal  habits  must  in- 
variably give  way,  in  all  the  manipulations  of  the  laboratory,  to  the  precise  care  of 
surgical  procedures  on  the  living  subject ;  and  it  is  desirable  that  even  in  the  matter  of 
clothing  similar  precautions  toward  cleanliness  and  protection  should  obtain. 

In  case  of  accidental  breakage  or  spilling  of  a  tube  containing  a  culture  upon  the 
floor,  table,  or  elsewhere,  as  much  as  possible  should  be  wiped  up  with  a  wet  rag  at  once 
and  the  rag  thrown  into  the  laboratory  disinfecting  jar,  to  be  burned  later ;  and  a  strong 
disinfectant  solution  should  be  freely  poured  over  the  contaminated  surface  and  allowed 
to  remain  for  a  half  hour,  after  which  it  may  be  wiped  up  with  a  mop  and  the  mop  de- 
stroyed. Clothing  thus  contaminated  should  be  boiled  or  baked ;  and  the  hands  should 
be  thoroughly  disinfected  and  washed  well  with  soap  and  water. 

Laboratory  Provisions. — Each  student  is  furnished  with  a  suitable  work-table,  with 
non-absorbent  top,  provided  with  water  and  gas  connections ;  for  general  use  are  pro- 
vided the  various  types  of  sterilizing  apparatus,  incubator  space,  serum 'inspissators, 
gas  generators,  material  for  culture  media,  chemical  tests,  staining  agents,  scales,  inocu- 
lating apparatus,  and  animals  for  inoculation.  Each  student  is  provided  with  an  indi- 
vidual locker  containing  the  following  pieces  of  apparatus,  .and  can  obtain  by  applica- 
tion the  less  frequently  needed  pieces  for  special  work: 

1  microscope  with  usual  accessories,  with  high-power  objective  and  bottle  of  im- 
mersion oil,  and  with  micrometer  ocular. 

6  dozen  culture  test-tubes. 

1  dozen  large  test-tubes  for  potato  cultures. 

1  large  double  dish  for  potato  and  plate  cultures. 

10  small  double  dishes  (Petri  dishes). 

1  straight  platinum  needle  with  glass  handle. 

1  looped  platinum  needle  with  glass  handle. 

Assorted  glass  rods  and  glass  tubing. 

1  50  c.c.  pipette. 

1  5  c.c.  pipette. 

2  glass  beakers. 

2  500  c.c.  Erlenmeyer  flasks. 

1  small  glass  funnel. 

1  large  glass  or  granite  funnel. 

1  large  granite  pan. 

1  granite  water-bath  pan  (bain  marie). 

1  granite  dipper,  500  c.c.  capacity. 

1  filtration  bucket. 

1  gas  stove. 


12  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

1  Bunsen  burner  with  wing  and  gauze  tops. 

Assorted  rubber  tubing. 

1  iron  tripod. 

1  iron  filter  stand,  three  rings. 

1  wooden  test-tube  rack. 

2  wire  cages  for  containing  test-tubes. 
1  hand  brush. 

bottle  brush. 

test-tube  brush. 

potato  knife. 

anaerobic  jar  and  attachments. 

dissecting  forceps,  scissors  and  scalpel  in'case 
6  bottles  with  stoppers  for  dropping,  for'special  reagents. 
6  reagent  bottles. 
12  staining  tubes  in  rack. 
1  slide  for  hanging-drop  culture. 
Staining  solutions,  reagents,  filter  paper,  wrapping  paper,  litmus  paper,  and  similar 

test  papers  and  solutions,  cheese-cloth,  etc. 

In  addition  to  the  above,  each  student  should  provide  himself,  for  personal  use  in 
the  laboratory,  a  suitable  apron  or  gown  of  washable  material,  soap,  a  number  of  rubber 


/ 

FIG.  i.  —  FORM  TYPES  OF  BACTERIA. 

/.    Coccaceae.      2.    Bacteriacese.     j.    Spirillacese.       ^t.    Mycobacteriacese.     j.  Chlamidobac- 

teriacese. 

finger  stalls,  cover-glass  forceps,  glass  slides  and  thin  cover-slips,  box  for  slides,  tube  of 
balsam  dissolved  in  xylol,  wax  pencil  for  temporary  labels  on  culture  tubes,  and  several 
hand  towels. 

Bacteria  belong  to  the  vegetable  kingdom  of  created  things,  constituting  one  group 
of  the  fungi,  or  non-chlorophyllous  protophytes.  They  are  also  spoken  of  as  the  schiz- 
omycetes  (sing.,  schizomyce  s)  ,  or  cleft  fungi,  or  fission-fungi,  because  among  this  group 
they  are  characterized  by  the  more  or  less  constant  and  common  method  of  multiplica- 
tion by  direct  cell  division,  as  contrasted  with  sporulation,  which  is  characteristic  of  the 
moulds,  and  with  gemmation,  which  is  encountered  among  the  yeasts.  While  this 
feature  is  essentially  a  basic  one,  it  is  not  absolutely  constant,  a  few  forms  usually,  and 
a  number  of  forms  under  special  conditions,  reproducing  by  spore  formati6n.  As  a 
group  they  are  probably  the  lowest  type  of  vegetables  and  bear  many  similarities  to  the 
lower  protozoa  (infusoria)  on  the  one  hand,  and  to  the  higher  types  of  the  fungi  and 
algae  on  the  other. 

They  exist  as  unicellular  organisms,  isolated  or  in  various  groupings  ;  are  spherical, 
oval,  rod-shaped,  or  curved  in  outline  ;  and  as  far  as  known  are  of  simple  structure,  in 
some  forms  only  presenting  as  special  organs  flagella,  by  which  motility  is  accomplished. 
For  convenience  they  may  be  divided  into  the  following  families:  Coccacece  (or  cocci),  of 


14  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

spherical  shape;  bacteriacece  (including  the  non-motile  bacteria  and  the  motile  bacilli 
and  pseudomonads) ,  unbranched  and  non-sheathed  rods;  mycobacteriacecz,  rod-shaped 
organisms  approaching  the  hyphomycetes  in  forming  mycelial-like  threads  and  some- 
times truly  branching;  spirillacea,  curved  rods;  and  chlamidobacteriacece,  filamentous 
bacteria  covered  with  more  or  less  definite  envelopes  (Fig.  1). 

For  further  division  of  these  families  reference  may  be  made  to  the  classification  in 
Lesson  IX  (vide  Chester,  Determinative  Bacteriology). 

Bacteria  occur  widely  in  nature,  both  as  free  and  parasitic  organisms,  scarcely  any 
substance  being  free  from  their  presence.  In  their  growth  they  approach  the  character 
of  the  protozoa  because  of  the  absence  of  chlorophyll  from  their  constitution,  rendering 
them  unfit  to  obtain  nutrition,  like  the  chlorophyllous  plants,  from  inorganic  substances  ; 
they  are  therefore  to  be  found  in  greatest  profusion  in  situations  where  organized  food  is 
available,  and  are  least  plentiful  where  such  food  is  not  obtainable  for  one  or  other  rea- 
son. The  vegetable  nature  of  bacteria  is  assumed  mainly  from  the  evident  close  relation- 
ship to  other  forms  of  protophytes,  often  seen  in  gradation  of  structural  and  functional 
characters ;  and  in  the  fact  that  at  least  in  a  number  of  the  bacteria,  if  not  all,  cellulose 
or  modifications  of  cellulose  have  been  demonstrated  as  a  more  or  less  important  con- 
stituent of  the  cell-wall.  Their  large  proportion  of  proteid  material,  as  well  as  many  of 
their  structural  and  functional  features,  also  ally  them  to  the  lower  animal  life ;  but 
from  a  preponderance  of  vegetable  features  they  are  generally  accepted  as  members  of 
the  vegetable  kingdom. 

Their  importance  to  the  physician  lies  in  their  frequent  parasitic  occurrence.  When 
found  growing  free  in  dead  organic  matter  they  are  spoken  of  as  saprophytes.  Of  the 
parasitic  forms  the  greater  number  are  productive  of  no  important  influences  deleterious 
to  the  well-being  of  the  host,  and  may  occasionally  be  of  positive  service;  such  are 
spoken  of  as  non-pathogenic  parasitic  bacteria.  However,  a  number  are  known  to  possess 
the  power  of  inducing  disease  in  the  body  of  the  individual  invaded  and  are  hence  spoken 
of  as  pathogenic  bacteria.  It  is  to  be  recognized,  however,  that  saprophytism  and  para- 
sitism may  in  turn  be  possible  in  numerous  instances  for  the  same  bacterium,  should 
circumstances  demand ;  and  while  an  organism  may  ordinarily  be  a  saprophyte,  it  may 
be  capable  of  living  as  a  parasite,  and  in  the  latter  condition  might  possibly  be  actively 
pathogenic.  Hence,  while  for  the  most  part  the  essentially  pathogenic  bacteria  demand 
the  physician's  attention,  there  are  more  reasons  than  that  of  general  interest  which  re- 
quire the  study  of  other  forms  as  well  as  of  these  latter. 

The  study  of  these  low  vegetables  is  known  as  Bacteriology  and  includes  not  only 
the  general  consideration  of  the  morphology  and  physiology  of  these  microorganisms, 
but  also  the  technique  of  observations  for  the  identification  of  the  organism  in  ques- 
tion, and  its  relations  to  disease. 


LESSON  II. 

OCCURRENCE  OF  BACTERIA  IN  NATURE;  THEIR 
DESTRUCTION  BY  LABORATORY  MEANS. 

Occurrence  of  Bacteria  in  Nature. — Bacteria  are  found  -very  -widely  distributed  in 
nature  both  as  saprophytes  and  as  parasites. 

The  wide  distribution  of  these  microorganisms  in  nature  can  readily  be  shown  by 
placing  in  material  free  from  bacteria  (sterile),  but  known  to  be  suited  to  their  nutrition, 
matter  containing  such  organisms  taken  from  a  variety  of  sources  such  as  may  suggest 
themselves.  It  is  due  to  this  almost  universal  presence  of  these  and  other  low  forms  of 
life  that  almost  every  organic  substance,  whether  liquid  or  solid,  is  so  apt  to  become 
destroyed  by  "rotting,"  especially  if  the  factors  of  temperature  and  moisture  of  the  sur- 
rounding medium  be  likewise  favorable  to  the  development  of  the  germs.  As  illustra- 
tive examples  the  following  experiments  may  be  suggested  and  may  be  added  to  at 
almost  any  length  at  the  will  of  the  inquirer. 

Exercise  i. — With  a  knife  blade  which  has  at  the  time  been  sterilized 
(cleansed)  by  careful  heating  in  the  smokeless  flame  of  a  Bunsen  burner  or 
alcohol  lamp  (the  blade  having  then  been  allowed  to  cool  to  a  convenient 
temperature  without  having  been  placed  where  it  is  likely  to  again  become 
contaminated),  some  of  the  superficial  cells  of  the  skin  of  the  hand  are 
scraped  from  the  surface  and  a  bit  of  the  scrapings  transferred  to  a  sterile 
tube  of  any  suitable  culture  medium,  as  nutrient  bouillon.  The  tube,  re- 
sealed,  is  to  be  placed  in  an  incubator  holding  a  uniform  temperature  of 
from  20°  C.  to  37°  C.  for  from  twenty-four  to  seventy-two  hours.  After  a 
time  the  presence  of  a  living  growth  may  be  inferred  by  the  clouding  of  the 
bouillon  or  by  the  formation  upon  the  surface  or  in  the  mass  of  solid  media 
of  "colonies,"  appearing  as  drops  or  films  of  a  colorless,  or  occasionally 
tinted,  material.  Microscopic  examination  will  confirm  the  statement  that 
such  material  is  made  up  of  countless  organisms  of  some  type.  Note  re- 
sults at  the  close  of  twenty-four,  forty-eight,  and  seventy-two  hours. 

Exercise  2. — In  a  like  manner  plant  scrapings  from  the  mucous  mem- 
brane of  the  mouth.  Note  results  at  the  close  of  twenty-four,  forty-eight, 
and  seventy-two  hours. 

Exercise  3. — Having  sterilized  the  platinum  loop  in  the  naked  flame  and 
allowed  it  to  cool,  a  drop  of  water  from  the  tap  is  similarly  introduced  into 
a  sterile  tube  of  nutrient  bouillon  or  other  medium,  placed  into  the  incu- 
»  16 


18  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

bator,  and  the  results  noted  at  the  close  of  twenty-four,  forty-eight,  and 
seventy-two  hours. 

Exercise  4. — In  a  similar  manner  a  bit  of  dust  from  the  floor  or  other 
exposed  surface  in  the  laboratory  is  obtained  by  drawing  the  sterilized 
platinum  needle  over  it,  which  is  then  brought  in  contact  with  a  prepared 
sterile  tube  of  some  nutrient  medium,  the  latter  placed  in  the  incubator, 
and  results  noted  at  the  close  of  twenty-four,  forty-eight,  and  seventy-two 
hours. 

Exercise  5. — Expose  for  fifteen  minutes,  to  the  atmosphere  of  the  room, 
a  dish  preparation  of  gelatine  or  agar;  close  the  dish  and  at  room  tempera- 
ture for  the  former,  or  at  incubator  temperature  for  the  latter,  note  at  the 
close  of  twenty-four,  forty-eight,  and  seventy-two  hours  the  results  of  the 
probable  inoculation  of  the  medium  from  the  air  of  the  room. 

STERILIZATION. 

By  this  term  is  meant  the  cleansing  of  any  substance  so  as  to  free  it  from  such  living 
organisms  as  may  exist  within  or  upon  it.  Just  as  it  is  necessary,  in  the  preparation  of  a 
garden  intended  for  the  cultivation  of  useful  vegetables,  to  free  the  soil  from  weeds 
which  may  interfere  with  the  growth  and  excellence  of  the  desired  plants,  so  it  is  essen- 
tial that  all  substances  upon  which  it  is  intended  to  grow  some  given  form  of  bacteria  be 
first  freed  from  all  contaminating  organisms.  So,  too,  lest  such  contamination  occur 
subsequently  and  more  or  less  continuously,  all  containers  and  such  other  apparatus  as 
in  the  necessary  manipulations  will  come  in  contact  with  the  nutrient  media  employed, 
must  likewise  be  subjected  to  sterilization.  Thus  alone  can  one  hope  to  so  isolate  his 
specimen  as  to  make  the  study  of  its  individual  characteristics  a  possibility  and  to  avoid 
a  hopeless  confusion  and  indistinguishable  mixture  with  any  number  of  other  types  of 
bacteria  and  other  protophytes. 

(A)  To  accomplish  this  end  heat  is  one  of  the  most  available  means  at  hand.  It  is 
employed  in  a  number  of  different  manners,  each  suitable  for  some  particular  substance 
or  for  some  particular  object  in  connection  with  the  general  purpose  of  sterilization. 

1.  Flaming. — This  consists  in  exposing  the  object  to  be  sterilized  to  the  naked, 
smokeless  flame  of  a  Bunsen  burner  or  of  an  alcohol  lamp  until  it  may  be  fairly  presumed 
that  any  living  matter  upon  its  surface  has  been  destroyed.  It  is  usually  practised  for 
the  rapid  cleansing  of  such  objects  as  the  platinum  inoculating  needle,  blades  of  knives 
(where  the  preservation  of  the  temper  of  the  blade  is  no  object),  glass  rods,  and  other 
metallic  or  glass  apparatus  of  suitable  size,  and  such  as  are  unlikely  to  be  injured  by 
the  exposure.  The  object  to  be  sterilized  should  be  held  in  the  upper,  hottest,  part  of 
the  flame,  the  time  of  exposure  being  usually  one  or  twe  minutes,  according  to  the  size  of 
the  surface  to  be  exposed  to  the  heat,  and  the  probable  degree  of  contamination  to  be  de- 
stroyed. With  metallic  objects,  like  the  platinum  needle,  exposure  is  usually  main- 
tained until  a  distinct  glow  of  the  metal  is  attained.  Having  presumably  thus  burned 
all  vestiges  of  life  from  the  object,  it  is  commonly  allowed  to  cool  to  a  harmless  tempera- 
ture before  being  further  used,  lest  contact  with  the  heated  surface  be  destructive  to 
such  substances  or  organisms  desired  to  be  preserved  with  which  the  sterile  object  must 
subsequently  be  brought  into  close  relation.  As  much  care  as  possible  should  be  taken 
to  prevent  contamination  of  the  sterilized  object  after  it  has  been  flamed  in  the 


20 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


moments  during  which  it  is  being  allowed  to  cool  for  use.     Thus  it  is  well  to  hold  the 
platinum  needle  perpendicularly,  point  downward,  so  as  to  expose  the  least  surface  to 
bacteria  settling  by  gravity  through  the  surrounding  air  (Figs.  2  and  3),  and  for  the 
same   reason   to   hold   a  knife  under  these   circumstances  in   a 
similar  position  or  horizontally  with  the  cutting  edge  downward. 

Exercise  6. — Two  tubes  of  sterile  nutrient  bouillon  or 
other  medium  are  provided.  A  platinum  inoculating 
needle  is  purposely  contaminated  by  being  thrust  into 
a  tube  of  decomposing  meat  solution  or  other  similar 
substance.  Without  further  precaution  it  is  thrust  into 
one  of  the  prepared  tubes  of  sterile  medium,  moved 
gently  in  it  for  a  moment  and  withdrawn,  and  the  tube 
resealed  with  the  usual  cotton  plug.  The  needle  is  now 
heated  throughout  its  entire  length  to  a  cherry  heat  and 
the  lower  portion  of  the  glass  handle  also  cautiously 
heated ;  after  which  the  needle  is  thrust  into  the  second 
tube  of  sterile  medium,  moved  about  therein  for  a 
moment  and  withdrawn,  and  the  tube  closed.  Note 
results  at  the  close  of  twenty-four,  forty-eight,  and 
seventy-two  hours  so  as  to  determine  the  efficiency  of 
the  flame  to  destroy  the  known  bacterial  contamination 
upon  the  needle. 

2.  Exposure  to  Dry  Oven  Heat. — The  dry-air  oven  is  em- 
ployed in  the  sterilization  of  such  articles  as  are  not  liable  to  be  destroyed  by  drying, 
but  which  do  not  demand  rapidity  of  cleansing,  or  are,  because  of  their  size  or  for  other 
reason,  not  easily  cleansed  by  exposure  to  the  direct  flame.      It  is  usually  used  in 
sterilizing  culture  tubes  and  their  cotton  plugs, 
for  the  sterilization  of  Petri  dishes,  flasks,  glass 
plates,  and  similar  objects.     The  articles  to  be 
sterilized  are  placed  in  the  oven,  either  inclosed 
in  some  suitable  receptacle,  as  in  wire  cages, 
metal  boxes,  or  folded  in  wrapping  paper  (as 
may  be  practised  with  Petri  dishes,  filtration 
bougies,   glass   rods,    pipettes,  etc.),  or  loose; 
and   heat   is   applied  in  the  most  convenient 
manner.     The  type  of  oven  commonly  used  in 
the  laboratory  is  a   double-walled  sheet-iron 
box,  covered   with   asbestos   card   to   prevent 
heat   loss,  and  provided  with  a  suitable  door. 
A  large  opening  at  the  bottom  of  the  outer  wall 

permits  the  hot  air  from  the  flame  to  enter  and  circulate  between  the  outer  and  inner 
walls,  and  a  series  of  smaller  openings  at  the  top  in  the  outer  wall,  guarded  by  a  slide 
valve,  allows  the  escape  of  the  hot  air  and  gas  after  circulation.  In  this  way  the  heat 
is  evenly  distributed  to  the  entire  inclosure.  A  section  of  such  hot -air  oven  is  repre- 
sented in  figure  4. 


FIG.  2.  —  POSITION 
IN  WHICH  INOCU- 
LATING NEEDLE 
is  HELD  WHILE 
COOLING. 


FIG.  3. — PROPER  POSITION  OF   HOLD- 
ING KNIFE  AFTER  FLAMING. 


22 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


While  there  is  undoubted  advantage  in  thus  evenly  distributing  the  heat  to  the 
different  parts  of  the  inclosed  space  of  such  an  oven,  it  is  not  absolutely  necessary,  and 
the  same  results  may  be  surely  obtained  by  the  employment  of  any  available  oven.  The 
oven  of  any  stove  may  be  used  for  the  purpose,  a  thermometer  being  introduced  to  es- 
tablish certainly  the  presence  of  the  requisite  degree  of  temperature ;  and  the  same  end 
may  be  attained  with  even  as  simple  an  apparatus  as  an  ordinary  tin  cracker  box  (with 
unsoldered  seams)  placed  upon  the  top  of  a  stove  or  other  source  of  heat,  or  in  an  old- 
fashioned  Dutch  oven. 

For  destruction  of  most  bacteria  by  dry  heat  a  temperature  of  140°-150°  C.  is  usu- 
ally regarded  as  essential,  although  many  adult  forms  are  killed  by  a  heat  much  less  than 
this.  The  spores  of  bacteria  are,  however,  more  resistant  than  the  adult  forms,  and  the 

degree  indicated  may  be  accepted  as  essential 
for  the  complete  destruction  of  all  forms. 
For  the  destruction  of  the  adult  varieties, 
exposure  of  from  twenty  to  thirty  minutes  to 
such  temperature  is  practically  always  lethal ; 
but  a  much  higher  temperature  is  required 
for  many  of  the  sporulating  forms,  and  occa- 
sionally even  prolonged  exposure  to  150°  C. 
will  fail  to  destroy  some  spores.  In  order  to 
obviate  such  difficulty,  Tyndall,  in  1877,  sug- 
gested the  so-called  "  fractional  sterilization  " 
(or  interrupted  sterilization)  in  connection 
with  this  mode  of  procedure  as  well  as  in 
case  of  steam  sterilization;  and  at  present 
this  is  usually  followed.  Such  method  de- 
mands an  exposure  of  the  object  to  be  steri- 
lized in  the  oven  to  a  temperature  of  140°- 
150°  C.,  for  from  twenty  to  thirty  minutes 
each  day  for  three  days,  the  intervals  between 
the  heatings  being  afforded  so  as  to  permit 
the  development  of  any  existing  spores  into 
adult  bacteria  and  consequent  loss  of  their 
powers  of  resistance  to  the  heat. 
FIG.  4. — SECTIONAL  VIEW  OF  HOT-AIR 

OVEN.  Exercise    7. — Select     six    contami- 

nated  tubes  from  previous  exercises. 

Pour  out  the  clouded  bouillon,  or  wash  out  with  ordinary  water  the  con- 
taminated solid  medium  into  the  disinfecting  jars  distributed  about  the 
laboratory,  taking  care  lest  the  substance  be  introduced  into  any  cuts  or 
abrasions  on  the  hands,  and  at  once  sterilizing  the  hands  by  washing  for  at 
least  five  minutes  in  a  one  in  two  thousand  solution  of  bichloride  of  mer- 
cury. Into  the  first  of  these  tubes,  without  further  preparation,  introduce  a 
few  cubic  centimeters  of  sterile  bouillon,  and  close  with  sterile  cotton  plug, 
as  usual.  Heat  a  second  tube  in  the  hot-air  oven,  in  which  a  temperature 
of  150°  C.  has  been  attained,  for  five  minutes ;  and  in  the  same  manner  there 
are  to  be  added  a  few  cubic  centimeters  of  sterile  bouillon,  and  the  tube 


24  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

again  closed  with  its  cotton  plug.  A  third  tube  is  heated  similarly  for  ten 
minutes,  bouillon  added,  and  the  tube  closed.  A  fourth  tube  is  heated  for 
twenty  minutes,  bouillon  added,  and  the  tube  closed.  The  fifth  tube  is 
heated  for  thirty  minutes  and  similarly  treated ;  and  the  last  tube  is  heated 
for  twenty  minutes  each  day  for  three  days,  to  the  same  temperature,  after 
which  sterile  bouillon  is  added  and  the  tube  closed  and  placed  in  the  incu- 
bator, which  should  also  have  been  done  with  the  former  tubes.  The  re- 
sults of  such  heating  in  each  case  should  be  noted  and  compared  with  the 
results  obtained  in  the  unclean  and  unheated  tube,  at  the  close  of  twenty  - 
four,  forty-eight,  and  seventy-two  hours. 

Exercise  8. — A  tube  containing  some  marked  growth  is  selected.  In- 
oculation is  performed  from  this  material  to  a  fresh  tube,  the  latter  closed 
and  placed  in  the  incubator.  The  original  tube  is  then  subjected  to  dry 
heat  for  five  minutes,  ten  minutes,  fifteen  minutes,  twenty  minutes,  twenty- 
five  minutes,  and  thirty  minutes.  At  the  close  of  each  interval  a  fresh  tube 
of  sterile  medium  is  inoculated  from  the  original  tube,  and  after  being 
closed  with  its  sterile  cotton  plug,  is  placed  in  the  incubator,  and  results  are 
noted  as  usual,  at  the  close  of  each  twenty-four  hours,  for  three  days.  That 
tube  in  which  growth  first  fails  to  occur  probably  corresponds  with  the  lethal 
exposure  to  the  temperature  to  which  the  tube  was  heated  (vide  Thermal 
Death-point) . 

3.  Boiling. — The  temperature  of  boiling  water  (100°  C.)  is  easily  destructive  of 
bacterial  life  if  long  enough  continued  or  if  the  conditions  for  penetration  of  the  heat  are 
favorable.     This  ease  of  penetration  is  present  best  when  the  bacteria  to  be  destroyed 
are  suspended  in  the  fluid  (rather  than  when  inclosed  in  fabric  or  other  solid)  which 
is  being  boiled,  the  hot  liquid  coming  into  close  contact  with  the  organisms  in  every 
part  and  on  every  side.    As  a  rule  it  may  be  said  that  sterilization  by  boiling  is  quite  as 
efficient  as  by  exposure  to  a  temperature  of  150°  C.  of  dry  heat.     Boiling  is  particularly 
useful  in  the  sterilization  of  articles  likely  to  be  destroyed  by  dry  heat,  as  clothing,  non- 
coagulable  nutrient  fluids,  glassware  which  may  be  cracked  by  any  irregularities  in  the 
application  of  the  oven  heat,  etc. 

Exercise  9. — Boil  water  for  five,  ten,  fifteen,  and  twenty  minutes,  and  at 
the  close  of  each  interval  add  several  drops  to  a  tube  of  sterile  medium; 
close,  place  in  incubator,  and  at  intervals  of  twenty -four  hours  for  three 
days  note  the  results  obtained.  Compare  with  a  tube  inoculated  with 
water  not  previously  boiled. 

4.  Steam  Sterilization. — Steam  heat  is  commonly  used  for  the  sterilization  of  such 
substances  as  are  likely  to  be  injured  by  heating  in  the  dry-air  oven,  and  which  cannot 
conveniently  be  boiled  or  sterilized  by  chemical  or  mechanical  means.     It  is  most  fre- 
quently used  in  connection  with  the  various  culture  media,  and  in  practical  medicine  and 
surgery  in  the  sterilization  of  dressings  for  wounds,  of  clothing  and  similar  substances.  The 
temperature  of  steam  unconfmed  by  the  lid  of  the  steam  chamber  is  approximately  85° 


26 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


C. ;  that  of  partially  confined  steam  (as  in  any  of  the  common  steam  sterilizers,  where  the 
lid  of  the  receptacle  is  closed,  but  not  tightly  clamped,  and  where  perhaps  there  is  in  ad- 
dition some  opening  in  the  lid  also  serving  as  a  means  of  moderate  escape  of  the  vapor) 
is  about  100°  C.  or  that  of  the  boiling  water;  while  the  temperature  of  steam  confined 
under  pressure,  as  in  the  specially  constructed  steam  boilers  known  as  steam  digesters  or 
autoclaves,  may  be  raised  considerably  above  that  of  boiling  water.  The  exact  degree 
in  the  latter  case  varies  with  the  degree  of  confinement,  whether  one  or  more  atmos- 
pheric pressures  above  normal,  and  with  the  amount  of  water  in  the  boiler  (the  less  water 
after  development  of  the  steam,  the  higher  the  temperature  for  a  given  application  of 
heat). 

Steam  as  a  sterilizing  agent  is  of  value  because  of  its  penetrative  power  as  com- 
pared with  dry  heat,  the  evenness  of  its  contact, 
and  steadiness  of  its  action.  Exposure  to  steam 
at  100°  C.  may  be  reckoned  as  of  equal  efficacy 
for  sterilization  as  dry  heat  at  150°C. ;  and 
ordinarily  it  is  the  custom  to  allow  the  same 
period  of  exposure  as  that  employed  in  the  use 
of  dry  heat,  twenty  to  thirty  minutes,  at  inter- 
vals of  twenty-four  hours,  upon  three  successive 
days.  If  the  substance  to  be  sterilized  is  not 
liable  to  injury  upon  prolonged  exposure,  it 
may  be  left  in  the  steam  at  100°  C.  for  an 
hour  with  a  fair  certainty  that  all  adult  bac- 
teria and  spores  will  at  the  end  of  that  time  be 
destroyed.  For  rapid  and  complete  steriliza- 
tion, however,  exposure  to  high  temperature  in 
steam  under  pressure  in  an  autoclave  is  usually 
practised,  a  temperature  of  120°  C.  and  pres- 
sure of  thirty  pounds  to  the  square  inch  being 
usually  employed,  an  exposure  of  from  fifteen 
to  twenty  minutes  being  generally  lethal  to 
both  adult  bacteria  and  their  spores. 

(a)  In  ordinary  steam  sterilization  some 
one  or  other  form  of  steam  boiler  is  made  use 
of,  in  which  is  attained  a  temperature  of  100°  C., 
or  under  special  arrangement  somewhat  less. 
Perhaps  the  simplest  type,  and  that  most  con- 
venient for  a  variety  of  purposes,  is  the  Koch  pattern  (Fig.  5),  consisting  of  a  rather 
tall,  heavily  tinned  sheet-iron  boiler,  with  copper  bottom,  arranged  upon  an  ordinary 
iron  tripod  or  upon  an  inclosed  iron  stand,  covered  with  asbestos  felt  to  prevent  the 
rapid  radiation  of  heat,  and  fitted  with  a  convenient  water  gauge  and  a  moderately 
close  fitting  cover  in  which  a  suitable  perforation  is  often  provided  for  the  accommoda- 
tion of  a  thermometer.  In  the  interior  above  the  water-level  is  arranged  a  false 
bottom  of  perforated  metal.  When  in  use,  water  is  put  in  the  boiler  so  as  to  cover  the 
bottom  for  four  or  five  inches,  but  not  enough  to  reach  the  false  bottom ;  heat  is  applied 
by  a  convenient  burner  and  the  water  brought  to  the  boiling-point.  The  articles  to  be 
sterilized  are  placed  in  the  steam  chamber,  resting  upon  the  false  bottom,  and  the 
cover  applied.  There  is  no  need  of  watching  the  temperature,  and  there  is  therefore  no 
occasion  for  a  thermometer,  since  the  temperature  cannot  rise  above  100°  C.  with  no 


FIG.  5. — KOCH  STEAM  STERILIZER. 


28 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


more  confinement  than  afforded  by  the  ordinarily  fitted  lid.  If  the  objects  subjected 
to  the  steam-bath  are  not  liable  to  injury  from  long  exposure,  the  sterilization  may  be 
accomplished  by  a  single  steaming  for  an  hour ;  but  ordinarily,  to  prevent  injury  and 
for  greater  certainty  of  result,  the  interrupted  method  of  Tyndall  is  followed,  the  arti- 
cles, after  subjection  to  heat  for  fifteen  or  twenty  minutes,  being  removed  from  the  bath, 
and  the  process  repeated  twice  at  intervals  of  twenty-four  hours. 

(b)  Another  form  of  steam  sterilizer  often  employed  is  the  Arnold  sterilizer  (Fig.  6), 
which  possesses  the  advantage  of  quick  generation  of  steam,  an  advantage  often  much 
appreciated  in  laboratory  work.  As  shown  in  the  diagram  in  section,  this  apparatus 
consists  of  a  steam  chamber  placed  over  a  thin  plate  boiler,  and  communicating 
with  the  latter  by  an  open  cylinder  through  which  the  steam  generated  in  the  boiler 

is  conducted  into  the  chamber.  A  water 
reservoir  surrounds  this  cylinder  and  com- 
municates with  the  boiler  beneath  by  means  of 
several  small  feed  tubes  for  the  supply  of 
water  as  it  is  vaporized.  In  the  bottom  of 
the  steam  chamber  a  perforated  false  bottom 
is  placed  for  the  accommodation  of  the  articles 
to  be  subjected  to  sterilization.  A  light  cover 
is  applied  to  the  steam  chamber,  loosely 
enough  to  allow  the  moderate  escape  of  ex- 
cess of  steam.  A  hood  of  metal  covers  the 
chamber,  in  the  interior  of  which  the  escaping 
steam  is  caught  and  condensed,  running  down 
its  sides  and  dropping  back  into  the  reservoir, 
to  be  used  over  again.  In  use,  the  reservoir 
should  have  water  poured  into  it  until  it 
stands  about  half  or  three-quarters  of  an  inch 
in  depth  over  the  bottom,  the  boiler  being 
thus  also  filled.  A  moderate-sized  burner  is 
placed  near  the  middle  of  the  bottom,  and  in 
a  few  minutes  a  clicking  sound  is  heard,  indi- 
cating the  boiling  of  the  water.  The  articles 
to  be  sterilized  are  now  placed  in  the  interior, 
the  cover  and  hood  adjusted,  and  after  a  few 
minutes  more  a  sterilizing  heat  has  been  at- 
tained. Exposure  in  this  apparatus  should 
be  the  same  as  in  the  case  of  any  other  form 

of  apparatus.  After  proper  exposure,  if  one  is  sterilizing  such  substances  as  fabric,  or 
tubes  plugged  with  cotton  stoppers,  it  is  well  in  this,  as  in  other  forms  of  steam  steril- 
izers, to  give  free  vent  to  the  steam  by  raising  the  cover  somewhat,  lest  the  contained 
steam,  in  cooling,  condense  upon  the  material  and  render  it  wet.  A  caution  should  be 
observed  in  the  employment  of  the  Arnold  sterilizer  as  to  the  heat  applied.  Should  the 
flame  be  fierce  and  the  temperature  employed  very  high,  it  is  not  safe  to  depend  upon 
the  maintenance  of  the  water-level  in  the  reservoir;  and  should  the  water  disappear, 
the  intricate  joints  of  the  apparatus  may  have  the  solder  melted,  especially  about  the 
small  feed  tubes,  the  damage  costing  considerable  inconvenience  and  expense  in  repair. 
If  used  with  ordinary  care  there  is  little  danger  of  such  accidents,  but  with  a  large  flame, 
as  from  a  gas  stove,  the  whole  apparatus  is  apt  to  become  overheated,  and  the  condensa- 


FIG.  6. — DIAGRAM  OF  ARNOLD  STEAM 
STERILIZER. 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


30 


^t         n  tfeam  '"  WS  n0t  take  place  With  certainty.  the  tevel  being  quickly 

tost  or  all  the  water  evaporated,  after  whieh  the  damage  is  done.  With  a  proper  flame 
the  reservoir  once  filled,  the  apparatus  requires  comparatively  little  attention-  but 
unless  students  are  careful  the  appliance  is  likely  to  be  more  costly  than  the  more  simple 

In  private  work  requiring  steam  sterilization  it  should  be  remembered  that  much 
simpler  apparatus  than  either  of  the  forms  mentioned  may  be  extemporized  whic™n 
accomphsh  all  that  a  reqmsrte,  the  expense  attendant  upon  these  set  forms  being  en- 
t,rely  unnecessary.  A  cheap  tin  bucket,  preferably  of  a  tall  shape,  should  be  obtafned, 

several  pieces  of  stone  or  brick  placed  in  the 
bottom,  and  a  piece  of  metal,  perforated  in  any 
convenient  manner,  arranged  upon  these  as 
supports,  thus  forming  a  raised  false  bottom. 
Water  is  put  into  the  bucket  nearly  to  the  level 
of  the  false  bottom,  the  articles  to  be  sterilized 
introduced,  and  the  cover  adjusted  and  heat 
applied.  Some  little  additional  care  is  requisite 
to  see  that  the  water  is  not  evaporated  before 
completion  of  the  process ;  but  otherwise  such  a 
crude  apparatus  presents  no  disadvantages  and 
will  serve  the  purposes  demanded  as  well  as 
apparatus  costing  a  number  of  dollars. 

(c)  For  rapid  and  thorough  sterilization  an 
autoclave  or   steam    digester  is   commonly  em- 
ployed.    In    principle    it    is    merely    a    heavy 
metallic  boiler,  capable   of    withstanding   con- 
siderable steam  pressure,  fitted  with  an  equally 
strong  cover  with  tight  fittings  and  clamps  for 
close  application  to  the  boiler.     There  is  usually 
a    steam    gauge    fitted    into  the  coyer,  and   a 
closed  tube  sunk  in  the  cover  for  the  reception 
of  a  thermometer;  a  safety  valve,  set  to  what- 
ever pressure  is  desired,  is  provided  in  the  cover 
or  elsewhere;  and  generally,  for  convenience,  a 
small  pet  valve,  closed  by  a  screw  plug,  is  also 
arranged.     In  order  to  conserve  the  heat  of  the 
flame  the  boiler  is  usually  set  upon  a  closed  stand 
of  heavy  sheet  iron,  the  inclosed  flame  not  being 
deviated  by  draughts,   and  little   heat   lost  by 
The  apparatus  can  readily  be  understood  by  reference  to  the  diagram  of 
a  section  (Fig.  7).     In  the  interior  there  is  a  false  bottom  or  gauze  bucket  or  other  con- 
:amer  for  the  reception  of  such  articles  as  are  to  be  subjected  to  the  process      There 
should  be  but  little  water  put  into  the  boiler  (the  bottom  being  covered  with  about  an 
>f  water),  and  a  strong  flame  applied.     At  first  the  cover  should  be  but  loosely 
applied  and  the  pet  valve  opened.     When  steam  begins  to  escape,  the  articles  to  be 
ihzed  are  to  be  placed  in  the  boiler,  the  cover  clamped  in  position,  the  pet  valve 
ed,  and  the  time  noted.     Usually,  an  exposure  of  fifteen  minutes  is  employed  after 
the  temperature  has  risen  to  120°  C.,  the  pressure  at  the  same  time  registered  by  the 
steam  gauge  being  about  thirty  pounds  to  the  square  inch.     The  heat  is  then  with- 


FIG 


7. — SECTIONAL  VIEW  OF  AUTO- 
CLAVE. 


32  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

drawn,  and  the  temperature  allowed  to  fall  to  somewhat  less  than  100°  C.,  and  the  pres- 
sure to  normal,  before  the  cover  is  removed  and  the  sterilized  materials  taken  from  the 
interior.  Should  one  attempt  to  open  the  cover  without  having  allowed  equalization  of 
pressure  to  have  taken  place,  fluids  in  the  interior,  as  culture  media  in  flasks  or  tubes, 
are  liable  to  bubble  violently,  wet  the  stoppers,  or  the  escaping  gases  may  force  them 
from  the  necks  of  the  flasks  or  tubes ;  and  the  operator,  if  careless,  is  liable  to  accidental 
scalding  from  the  rush  of  steam  from  the  apparatus.  One  such  exposure  is  easily  equal 
to  the  three  exposures  in  the  ordinary  method  of  operation,  but  it  should  be  recalled  that 
the  high  temperature  may  be  unsuited  to  some  materials. 

Exercise  10. — Repeat  exercise  8  in  duplicate,  employing,  instead  of  dry 
heat,  for  one  set  of  tubes  the  ordinary  fractional  sterilization  in  steam  with- 
out pressure,  and  for  a  second  set  sterilization  in  the  autoclave,  tempera- 
ture 120°  C.  and  a  pressure  of  thirty  pounds.  In  the  first  set  allow  one  in- 
fected tube  to  be  exposed  to  the  full  process,  one  to  but  two  exposures,  one  to 
a  single  exposure  of  twenty  minutes,  one  to  a  single  exposure  of  one  hour.  In 
the  second  set  let  one  tube  be  exposed  to  the  full  twenty  minutes'  heating, 
one  to  steam  for  fifteen  minutes,  one  ten  minutes,  and  one  five  minutes. 
Place  all  tubes  in  the  incubator  and  note  results  at  the  close  of  twenty-four, 
forty-eight,  and  seventy-two  hours.  The  tube  in  each  case  with  least  expo- 
sure showing  no  occurrence  of  growth  marks  the  lethal  exposure  of  the  given 
germ  to  the  mode  of  procedure  employed. 

In  determination  of  the  thermal  death-point  of  a  given  microorganism,  material  de- 
void of  spores  is  selected  (this  determined  by  microscopic  examination  or  by  the  success 
of  inoculations  from  cultures  which  have  been  exposed  to  a  temperature  of  80°  C.  for  ten 
minutes) .  This  material  may  be  either  diffused  in  a  sterile  fluid  in  tubes  or  smeared  dry 
on  sterile  glass  slides;  each  specimen  is  then  placed  in  one  of  a  series  of  ovens  the  tem- 
perature of  which  ranges  several  degrees  apart  through  a  number  of  degrees,  as  50°,  52°, 
54°,  56°,  58°,  and  60°  C.  At  intervals  inoculations  are  made  from  each  specimen.  The 
shortest  exposure  to  the  lowest  degree  of  heat  after  which  such  inoculations  fail  of 
growth  represents  the  temperature  lethal  to  the  bacterium.  In  the  record  note  should 
be  made  of  whether  the  organism  was  in  moist  or  dry  condition,  as  well  as  the  degree  of 
temperature  and  the  duration  of  exposure. 

Exercise  1 1. — In  order  to  observe  the  relative  value  of  steaming  without 
pressure  and  under  pressure,  some  infected  material,  as  a  fresh  surgical 
dressing  from  some  suppurating  wound  or  bits  of  sterile  threads  dipped  in 
a  bouillon  culture  of  some  known  organism,  as  the  colon  bacillus,  is  care- 
fully wrapped  in  a  known  number  of  thicknesses  of  a  towel.  One  such 
preparation  is  to  be  placed  in  the  autoclave  and  subjected  to  the  usual 
steam  exposure ;  the  other  is  placed  in  the  ordinary  steam  sterilizer  for  the 
same  period.  As  soon  as  removed,  inoculations  are  to  be  made  from  each 
of  these  preparations  to  fresh  tubes  of  nutrient  medium,  incubated,  and  the 
results  noted  as  usual  at  the  close  of  twenty-four,  forty -eight,  and  seventy- 


34  LABORATORY  EXERCISES  IN  BACTERIOLOGY, 

two  hours.     Should  both  prove  sterile,  the  experiment  is  to  be  repeated 
with  briefer  period  of  exposure. 

5.  Pasteurization. — This  term  is  applied  to  a  partial  sterilization  accomplished  by 
exposure  to  a  comparatively  low  temperature  (60°-90°  C.)  for  a  period  of  time  ranging, 
under  varying  circumstances,  from  fifteen  minutes  to  an  hour.  While  in  the  briefer 
exposures  to  the  lower  ranges  of  temperature  the  sterilization  is  usually  but  partial,  with 
longer  exposures  to  temperatures  somewhat  less  than  boiling  heat,  especially  steam  heat, 
and  particularly  when  the  procedure  is  repeated  from  time  to  time,  the  destruction  of 
bacteria  present  may  be  complete.  In  its  ordinary  sense  the  process  consists  of  steam- 
ing the  material  in  hand  at  a  temperature  of  60°  C.  or  over  for  fifteen  to  thirty  minutes ; 
it  is  especially  employed  for  the  preservation  of  milk,  this  exposure  being  sufficient  to 
destroy  at  least  most  of  the  pathogenic  and  souring  bacteria  which  have  been  present  in 
the  sample,  but  insufficient  to  cause  any  material  physical  change  in  the  milk.  The 
procedure  in  this  connection  was  instituted  to  avoid  the  changes  which  occur  in  milk 
upon  complete  sterilization  at  higher  temperatures  and  which  render  it  objectionable 
for  infant  feeding.  Essentially  the  same  process,  however,  had  long  been  employed  in 
the  preparation  of  clear  solidified  blood-serum,  although  in  the  latter  case  the  higher 
temperatures  (ranging  from  70°  to  85°  C.,  and  even  higher,  but  not  to  the  boiling-point), 
longer  exposures,  and  repetition  of  the  method  (one  hour  daily  for  a  week)  had  been 
practised.  Originally,  in  the  preparation  of  blood-serum  an  ordinary  steam-bath  was 
used;  but  for  years  dry  heat  in  a  chamber  inclosed  in  a  water-bath  (serum  inspissator) 
has  been  employed.  One  may  therefore  look  upon  Pasteurization  as  merely  the  appli- 
cation of  either  steam  or  dry  heat  at  a  low  temperature  for  partial  or  complete  steriliza- 
tion, such  temperature  more  or  less  completely  destroying  contained  bacteria  without 
causing  material  alteration  in  the  constitution  of  the  substance  handled. 

A  number  of  special  devices  are  used  for  Pasteurization,  especially  in  the  preparation 
of  milk  for  infants'  food ;  but  the  same  result  may  be  accomplished  by  heating  the  milk 
or  other  substance  in  freely  streaming  steam  (temperature  approximately  80-85°  C.), 
or  in  a  water-bath,  for  fifteen  to  twenty  minutes. 

Exercise  12. — Into  each  of  three  sterile  test-tubes  a  few  cubic  centi- 
meters of  sweet  milk  are  introduced  and  the  tubes  closed  with  sterile  cotton 
plugs.  One  is  at  once  placed  in  the  incubator.  The  second  is  Pasteurized 
in  streaming  steam  for  twenty  minutes.  With  a  sterile  pipette  the  student 
should  withdraw  a  few  drops  from  the  latter  to  observe  the  preservation  of 
the  normal  taste  of  the  sample,  and  should  note  also  the  preservation  of  the 
normal  color  of  the  specimen.  The  third  tube  is  introduced  into  the  auto- 
clave and  heated  to  120°  C.  for  fifteen  minutes.  On  withdrawal  note 
should  be  made  of  the  yellowish  or  brownish  alteration  in  color  usually  pro- 
duced, and  with  a  sterile  pipette  a  few  drops  are  to  be  withdrawn  in  order 
to  observe  the  change  in  taste  also  caused  by  the  heat  exposure.  The  steril- 
ized and  Pasteurized  tubes  are  now  to  be  placed  in  the  incubator  with 
the  first  tube  and  daily  note,  for  at  least  three  days,  made  of  each  as  to 
coagulation,  color,  reaction  to  litmus,  visible  growth,  etc.,  to  determine  the 
differences  in  effect  of  the  two  processes  upon  the  preservation  of  the  milk. 


36  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

(B)  Sterilization  by  Filtration.— The  principle  of  separation  of  solid  admixtures 
from  fluid  media  by  means  of  nitration  through  porous  substances  has  several  applica- 
tions in  bacteriology  in  the  matter  of  sterilization. 

(a)  While  bacteria  are,  of  course,  of  much  smaller  size  than  the  spaces  in  ordinary 
filter  paper  or  the  meshes  of  a  cotton  plug  or  fabric,  without  the  force  of  currents  of 
liquid  to  carry  them  they  are  unable  to  pass,  save  by  continuous  growth,  through  the 
intricate  spaces  of  a  moderately  thick  layer  of  the  paper  or  cotton ;  and  plugs  of  cotton 
or  several  thicknesses  of  paper  or  fabric  are  therefore  commonly  utilized,  after  thorough 
sterilization,  as  a  means  of  protection  to  the  interior  of  flasks,  tubes,  etc.,  from  the  en- 
trance of  bacteria  from  the  surrounding  air,  although  permitting  fairly  free  ingress  and 
egress  to  the  atmosphere  itself.     Moreover,  if  such  material  be  kept  dry,  and  free  from 
soiling  with  any  of  the  nutrient  substances  contained  in  the  flasks  or  tubes,  there  is  no 
opportunity  for  the  penetration  by  growth  of  any  organisms  settling  upon  their  exposed 
portions,  and  the  protection  from  external  contamination  thus  afforded  is  practically 
complete. 

Exercise  13. — From  a  small  glass  tube  blow  a  bulb  as  exhibited  in  figure 
8.  Plug  each  end  with  a  bit  of  cotton  and  sterilize  in  the  usual  manner. 

Introduce  into  the  bulb  a  small  amount 
of  melted  agar  or  gelatine  and  again 
sterilize  the  whole.  Now  attach  the 
apparatus  to  a  suction  pump  and  for 
several  minutes  draw  through  the  tube 
and  over  the  medium  contained  in  the 
bulb  air  which  has  been  filtered  through 
FIG.  8.  the  cotton  plug  in  the  exposed  orifice  of 

the  tube.      Repeat    the  same   with   a 

second  tube  similarly  arranged,  but  not  having  a  cotton  plug  in  the  end 
of  the  tube,  the  air  drawn  through  the  tube  being  unfiltered.  Place  both 
tubes  in  the  incubator  and  at  the  end  of  the  first,  second,  and  third  day 
note  the  presence  or  absence  of  growth  in  each. 

(b)  Filtration  through  various  porous  substances  has  commonly  been  used  in  the 
crude  purification  of  water  and  other  liquids ;  and  the  same  principle  is  employed  in  the 
laboratory  for  sterilization  of  liquids  from  bacterial  contamination  when  the  ordinary 
methods  of  heating  or  chemical  purification  are  not  to  be  permitted  because  of  the  im- 
portant physical  changes  liable  to  be  produced  in  the  liquids  subjected  to  such  processes. 
Filtration  through  paper,  cotton,  or  other  coarsely  porous  substances  is,  however,  of 
little  or  no  value,  the  bacteria  in  the  fluids  being  readily  conveyed  with  the  currents  of 
the  liquid  through  the  spaces  of  the  filter;  and  specially  prepared  filters  made  of  un- 
glazed  porcelain  are  commonly  employed  for  the  purpose.     The  Pasteur-Chamberland 
filter,  widely  used  for  the  filtration  of  water  for  drinking,  is  an  excellent  type.     Among 
various  models,  one  of  the  most  simple  and  efficient  is  that  of  Kitasato,  a  view  of 
which  is  shown  in  the  accompanying  diagram  (Fig.  9).     Such  porcelain  filters  may  be 
utilized  in  the  sterilization  of  various  liquid  culture  media,  of  water,  or  in  the  separation 
of  bacteria  from  infected  media  when  it  is  desired  to  obtain  the  various  chemical  pro- 
ducts of  bacterial  activity  free  from  the  bacteria  themselves.     When  used,  inasmuch  as 


38 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


the  liquids,  particularly  solutions  of  organic  matter,  pass  but  slowly  through  the  rather 
compact  porcelain,  it  is  commonly  necessary  to  attach  to  the  receiving  flask  some  form 
of  vacuum  pump,  an  ordinary  water  pump  connected  with  a  neighboring  faucet  being 
usually  used.  Care  should  be  exercised  to  have  the  connection  between  the  pump  and 
the  receiver  of  heavy,  non-collapsible  tubing  and  so  arranged  that  there  is  no  danger  of 
reversal  of  the  flow  of  water  so  as  to  enter  the  receiver.  Before  setting  up  such  filtration 
apparatus  it  is  essential  that  the  various  parts  be  sterilized.  Those  parts  made  of  glass, 
as  the  heavy  receiving  flask  and  funnel,  having  been  well  washed,  are  to  be  plugged  with 
cotton  and  baked  in  the  ordinary  manner  or  sterilized  in  the  autoclave.  The  rubber 
connection  and  the  perforated  rubber  stopper  should  be  soaked  in  a  solution  of  some 
suitable  disinfectant  (as  1 :  1000  solution  of  mercuric  chloride,  or  a  three  or  four  per  cent. 


FIG.  9. — KITASATO  FILTER. 


FIG.  10. — FILTER  REVERSED  IN  A  PERCO- 
LATOR FILLED  WITH  WATER  IN  ORDER 
TO  WASH  BACK  SOLID  PARTICLES  LODGED 
IN  ITS  WALL. 


solution  of  carbolic  acid)  and  well  rinsed  in  sterile  water.  The  porcelain  bougie  should 
have  been  wrapped  in  paper,  the  open  end  of  the  bougie  being  marked  on  the  outside, 
and  carefully  sterilized  in  the  autoclave.  All  of  the  parts  having  received  such  atten- 
tion, the  funnel  is  adjusted  in  the  perforated  rubber  stopper,  the  connecting  rubber  tube 
fitted  to  the  lower  end  of  the  funnel ;  and  the  open  end  of  the  bougie  being  exposed  from 
its  paper  cover  is  adjusted  in  the  lower  end  of  the  rubber  connecting  tube,  which  is  bound 
to  the  latter  and  to  the  lower  end  of  the  funnel  by  several  ties  of  sterile  copper  wire. 
The  bougie  is  then  freed  from  its  wrapping  paper  and  placed  in  the  receiving  flask  and 
the  rubber  stopper  tightly  fixed  in  place.  The  pump  connection  is  then  made  with  the 
side  tube  of  the  receiving  flask,  the  fluid  to  be  sterilized  introduced  into  the  funnel,  and 
the  pump  cautiously  started.  At  best  filtration  is  a  slow  process,  and  if  proceeding  at 
all  satisfactorily  should  not  be  interfered  with,  since  the  least  extra  force  of  current 


40  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

through  the  porcelain  may  be  capable  of  conveying  some  of  the  bacteria  through  the 
filter.  Moreover,  after  a  time,  with  any  degree  of  forced  filtration  there  is  danger  that 
the  smaller  forms  of  bacteria  will  slowly  penetrate  through  the  porcelain.  For  this  rea- 
son the  value  of  this  form  of  sterilization  is  limited  and  to  be  recommended  only  when 
other  methods  are  not  available  for  the  purpose  desired.  In  the  selection  of  the  filter 
care  should  be  exercised  that  the  porcelain  is  of  even  and  compact  composition,  and  of 
considerable  thickness  where  the  amount  of  fluid  to  be  filtered  is  at  all  large.  After  a 
filter  has  been  used  its  further  value  for  sterilization  is  often  doubtful,  for  several  reasons. 
It  may,  it  is  true,  be  cleansed  by  reversing  its  position  in  the  apparatus  (Fig.  10)  and 
drawing  through  it  a  large  amount  of  clean  water,  thus  washing  back  the  microbes  and 
other  solid  particles  lodged  in  its  interstices ;  after  which  it  is  to  be  resterilized  in  the 
usual  manner  in  the  autoclave.  Such  cleansing  is,  however,  in  many  instances  of  little 
practical  efficacy,  and  the  permeability  of  the  filter  in  consequence  diminished  even  if 
the  foreign  particles  are  of  no  vitality  after  exposure  to  the  heat  of  the  autoclave. 

Exercise  14. — Having  prepared  and  set  up  a  Kitasato  or  other  filter  as 
above  outlined,  filter  some  tainted  meat  solution  into  the  receiving  flask. 
Let  each  student  then  inoculate  a  fresh  tube  of  sterile  nutrient  medium 
from  the  filtrate,  place  the  inoculated  tube  in  the  incubator,  and  note  re- 
sults as  usual  at  the  end  of  the  first,  second,  and  third  days  in  order  to  de- 
termine the  efficiency  of  the  process  as  a  means  of  sterilization. 

Exercise  15. — Repeat  the  above  exercise,  employing  some  large,  known 
bacterium,  as  the  Bacillus  subtilis  in  a  hay  infusion,  as  the  material  for  in- 
oculation ;  and  in  the  same  way  note  results  after  inoculation  of  the  sterile 
nutrient  medium  with  such  material. 

(C)  Sterilization  by  Means  of  Chemical  Agents. — Various  chemicals  in  solution  or 
in  vapor  are  commonly  employed  for  the  purpose  of  bacterial  destruction  or  for  retarda- 
tion of  their  growth,  the  terms  disinfectant  and  germicide  being  used  to  indicate  such 
substances  as  are  capable  of  destroying  microbic  life,  and  antiseptic  to  indicate  such  as 
are  merely  capable  of  restraining  the  growth  or  other  functions  of  bacteria  without 
actually  killing  them.  The  confidence  which  was  formerly  reposed  in  many  of  the  so- 
called  disinfectants  has  in  recent  times  been  considerably  disturbed  by  the  discovery  that 
in  many  instances  the  bacteria  apparently  killed  are  really  living,  and  can  by  proper 
procedures  be  caused  to  resume  their  vital  activities.  It  was  formerly  supposed  that 
such  substances  as  mercuric  chloride,  which  may  be  taken  as  a  type  of  the  disinfectants, 
were  directly  lethal  by  mere  contact  with  the  germ,  the  chemical  substance  itself  not 
being  modified  by  such  contact.  Recent  studies,  however,  indicate  that  true  chemical 
compounds  are  formed  from  organic  materials  and  the  metallic  base  of  the  salt,  spoken  of 
collectively  as  mercurial  albuminates,  the  haloid  element  being  evolved  in  free  state  and 
itself  further  combining.  Such  destruction  of  the  disinfectant  and  combination  of  its 
elements  must,  of  course,  render  inert  for  further  use  in  sterilization  just  such  propor- 
tion of  the  salt  as  has  entered  into  the  chemical  change ;  and  the  albuminate,  thus 
formed  in  greater  or  less  amount,  acts  as  a  protective  covering  around  the  individual 
germs  and  upon  the  exterior  of  any  mass  which  should  be  penetrated  by  the  disinfectant 
solution.  This  coating  may  effectively  protect  the  individual  germs  against  further  ac- 
tivity of  the  disinfectant,  and,  although  mechanically  preventing  in  greater  or  less 
measure  the  manifestation  of  their  vital  phenomena,  may  permit  the  maintenance  of 


42  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

life.  It  has  been  shown  that  solution  of  such  covering  from  the  exterior  of  the  bacteria, 
by  means  of  some  chemical  solvent,  as  a  weak  solution  of  ammonium  sulphide,  may  be 
followed  by  resumption  of  the  life  activities  of  the  germs  if  they  be  transferred  to  a  favor- 
able surrounding.  Considerable  revision  of  opinion,  particularly  in  connection  with  the 
inorganic  disinfectants,  as  to  their  relative  sterilizing  value  has  thus  been  en- 
forced. At  present  it  is  believed  that  the  organic  disinfectants,  as  carbolic  acid,  and  the 
haloid  elements,  chlorine,  bromine,  and  iodine,  are  less  liable  to  give  rise  to  such  organic 
combinations  than  the  formerly  esteemed  metallic  salts,  as  those  of  mercury,  silver, 
copper,  and  iron ;  and  the  former  are  therefore  more  generally  commended  than  formerly 
for  the  purposes  of  sterilization.  The  objection  mentioned  concerning  bichloride  of 
mercury  obtains  in  varying  degree  with  the  other  disinfectant  salts  of  mercury  and  salts 
of  other  metallic  bases;  but  it  should  be  recalled  that  this  objection  may  be  largely 
obviated,  if  in  their  use  in  disinfection  one  remembers  that  the  process  is  essentially  a 
quantitative  one  and  employs  a  considerable  excess  of  the  disinfectant  solution  and 
allows  a  long  action  period  for  thorough  contact  of  the  chemical  with  the  bacteria  in  the 
mass  or  liquid  sought  to  be  sterilized.  In  this  connection  it  may  be  insisted  upon  as  a 
second  and  absolute  requisite  in  the  practice  of  disinfection  by  chemical  means,  that 
there  must  be  intimacy  of  contact  between  the  disinfecting  substance  and  the  bacteria  to  be 
destroyed,  and  that  such  contact  must  be  maintained  for  at  least  a  known  minimum  of 
time.  There  may  thus  be  formulated  as  a  rule  for  chemical  disinfection  that  in  all  such 
usage  there  should  be  employed  a  free  excess  of  the  disinfectant,  that  it  should  be  caused 
to  penetrate  and  diffuse  through  the  mass  or  liquid  subjected  to  its  action  so  as  to  be 
brought  in  intimate  contact  with  the  contained  bacteria,  and  that  it  should  be  allowed 
a  sufficient  period  of  time  in  which  to  accomplish  the  destruction  of  the  bacteria. 

In  the  use  of  these  chemical  agents  in  liquid  form,  they  are  almost  invariably  found 
more  efficient  if  moderately  heated  (40°  C.,  or  above).  They  are  not  comparable  to  the 
action  of  heat  and  are  to  be  used  only  when  every  form  of  heating  is  unavailable  for  one  or 
other  reason.  Among  the  substances  for  the  sterilization  of  which  the  chemical  disinfec- 
tants are  usually  selected,  may  be  mentioned  such  pieces  of  glassware  and  laboratory 
apparatus  which  on  account  of  size  cannot  be  introduced  into  the  sterilizing  ovens  or 
steam  chambers,  material  like  rubber  goods,  which  would  be  injured  in  the  heat,  refuse 
of  various  kinds,  as  the  rejected  contents  of  infected  tubes  (which  should  after  collection 
in  such  disinfectant  solution  be  burned  to  render  its  destruction  certain),  the  clothing 
and  dejecta  of  patients  affected  by  infectious  diseases,  and  a  variety  of  similar  sub- 
stances. 

Disinfectant  Solutions. — Of  the  various  disinfectant  solutions,  in  spite  of  the  objec- 
tion above  mentioned,  that  of  mercuric  chloride  (1 :  1000  or  1 :  2000)  has  been  and  con- 
tinues to  be  the  most  universally  employed.  Efforts  to  prevent  the  formation  of  the 
organic  compounds  of  mercury  have  been  made  by  the  addition  of  small  proportions  of 
such  materials  as  hydrochloric  acid,  sodium  or  ammonium  chloride,  or  tartaric  acid  to 
the  solution;  but  it  is  doubtful  whether  any  of  these,  save  the  last,  serves  any  efficient 
purpose  or  indeed  does  not  hasten  the  active  decomposition  of  the  mercurial  salt.  If 
used  in  free  excess  over  the  amount  of  material  subjected  to  it  for  disinfection,  the  fol- 
lowing stock  solution  offers  a  convenient  and  efficient  medium  for  laboratory  use : 

Mercuric  chloride, 200 

Tartaric  acid, 5°° 

Distilled  water  to 2000 

Ten  cubic  centimeters  of  the  above  to  a  liter  of  water  make  a  solution  of  1 :  1000 


44  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

strength.  In  use  this  solution,  as  all  others  of  the  chemical  disinfectant  solutions, 
should  be  allowed,  for  action,  from  half  an  hour  to  any  longer  period  which  can  profita- 
bly be  permitted,  and  should  always  be  used  in  great  excess  of  volume.  All  waste  and 
useless  materials  thus  sterilized  should  be  subsequently  burned  to  obviate  the  danger  of 
failure  of  sterilization ;  and  any  articles  to  be  further  used  should  be  well  rinsed  in  sterile 
water  before  being  employed. 

Among  the  other  salts  of  mercury  the  iodide  and  the  nitrate,  in  practically  the  same 
proportions  as  the  chloride,  may  also  be  employed  in  disinfection. 

Carbolic  acid  is  probably  superior  to  any  of  the  metallic  salts  in  the  certainty  of  its 
disinfectant  action,  but  is  slower  and  should  be  kept  in  contact  with  the  objects  to  be 
sterilized  for  periods  ranging  from  an  hour  to  twenty-four  hours  in  length.  It  is  usually 
used  in  solutions  of  two  and  a  half  to  five  per  cent,  strength.  The  addition  of  an  equal 
proportion  of  hydrochloric  acid  to  the  solution  materially  increases  its  activity,  and 
forms  one  of  the  most  valuable  laboratory  disinfectants.  It  also  forms  an  inert  com- 
pound with  organic  substances  with  which  it  comes  in  contact,  but  to  a  considerably  less 
degree  than  the  metallic  salts,  to  which  it  is  therefore  superior. 

Formaldehyde  possesses  valuable  germicidal  action  if  used  in  solutions  of  from  ten 
parts  of  the  commercial  article  (a  forty  per  cent,  watery  solution  of  formaldehyde  gas) 
to  1000  parts  of  water,  to  forty  parts  to  1000  of  water.  It  has  little  penetrative  power 
into  albuminous  substances,  however,  and  must  be  granted  considerable  time  for  its  com- 
plete action.  Its  odor  and  irritant  properties  prevent  its  general  use. 

Chlorinated  lime  is  one  of  the  most  valuable  disinfectants,  in  fresh  solution,  which 
we  possess,  depending  upon  the  free  chlorine  evolved  by  its  decomposition.  It  should 
be  used  in  solutions  of  from  one  to  three  or  four  per  cent,  strength,  made  from  the  fresh 
commercial  substance  as  required  for  use,  as  it  readily  decomposes.  Articles  to  be  ster- 
ilized should  be  kept  in  contact  with  an  excess  of  such  solution  for  at  least  an  hour;  and, 
as  is  always  required,  should  be  subsequently  rinsed  in  sterile  water  if  intended  for 
further  use.  It  is  an  excellent  disinfectant  in  the  sick-room  for  sterilization  of  clothing 
or  dejecta,  and  may  be  profitably  used  in  the  laboratory  waste  jars  for  preliminary  disin- 
fection of  the  matter  therein  collected  for  combustion.  Ordinary  lime  in  strong  (twenty 
per  cent.)  solution  (milk  of  lime  or  whitewash)  is  an  excellent  crude  disinfectant,  espe- 
cially useful  in  the  purification  of  dejecta  from  subjects  of  such  infections  as  typhoid 
fever  or  cholera. 

Copperas  (ferrous  sulphate),  commonly  used  as  a  disinfectant  in  urinals  and  cess- 
pools, is  of  comparatively  feeble  germicidal  value,  but  possesses  considerable  power  as  a 
deodorant.  Potassium  permanganate,  frequently  employed  in  one  of  the  steps  of  sur- 
gical disinfection,  is  of  little  value,  even  in  comparatively  strong  solution. 

For  discussion  of  the  numerous  other  agents  employed  in  disinfectant  solution, 
reference  should  be  made  to  the  various  text-books  upon  bacteriology. 

Antiseptic  Solutions. — Of  the  antiseptics,  borax,  boracic  acid,  iodoform  (from  the 
iodine  set  free  from  its  gradual  decomposition),  many  of  the  essential  oils  (as  of  cinna- 
mon, cloves,  thyme,  mint,  and  other  plants) ,  as  well  as  weak  solutions  of  the  disinfectants, 
may  be  mentioned.  Oil  of  mustard  is  both  antiseptic  and  deodorant,  but  is  too  irritant 
for  common  use ;  the  dry  mustard  meal  is  often  used  to  remove  odors  from  the  hands 
after  performance  of  post-mortem  operations  and  similar  work.  The  retardation  of  de- 
velopment of  any  germ  by  such  substances  is  not  the  only  phenomenon  of  antisepsis. 
In  addition  to  or  in  place  of  prevention  of  growth,  the  presence  of  such  substances  may 
modify  the  mode  of  multiplication  (perhaps  inducing  sporulation),  the  physical  charac- 
ters of  the  resultant  colonies  (as  in  shape  or  color),  or  the  pathogenic  virulence  or  other 


46  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

biologic  properties  of  the  bacteria  studied.  However,  the  modification  in  the  rate  of 
development  is  generally  taken  as  an  index  of  the  effect  of  all  such  antiseptic  influence. 

In  the  production  of  antisepsis  it  is  supposed  that  the  antiseptic  substance  is  con- 
tinuous in  its  presence  and  proportion  in  the  medium,  although  upon  the  least  reflection 
this  must  be  matter  of  question.  Were  such  substance  able  to  prevent  or  modify  growth 
by  its  mere  presence,  its  continuous  presence  thus  being  unmodified  must  result  in  the 
indefinite  prevention  of  development  and  the  eventual  natural  disappearance  of  the 
germs.  It  is  probable  that  the  activity  of  antiseptics  must  be  regarded,  just  as  that  of 
the  disinfectants,  not  as  a  katalytic  one,  but  as  a  process  of  quantitative  changes ;  and 
that  retardation  of  development  persists  only  until  a  certain  amount  of  the  antiseptic 
present  has  been  rendered  inert  by  combination  or  dissociation.  This  accomplished, 
such  of  the  bacteria  present  as  are  not  actually  destroyed  are  no  longer  interfered  with, 
but  develop  in  their  original  and  usual  manner  and  with  their  natural  characteristics. 
Further  study  is  required  for  the  establishment  of  the  truth  of  a  number  of  problems 
connected  with  the  subject. 

Determination  of  Disinfectant  Values. — The  greatest  dilution  of  a  given  sub- 
stance placed  in  excess  in  contact  with  a  given  bacterium  for  the  shortest  action  period 
(time  of  exposure)  causing  the  destruction  of  that  organism  is  spoken  of  as  the  disin- 
fectant value  of  that  substance  for  the  germ  employed  in  the  test.  Thus,  one  may  say 
of  bichloride  of  mercury  that  it  is  disinfectant  in  1 :  1000  solution  in  five  minutes  for  the 
anthrax  organism  in  its  vegetative  form,  although  its  spores  may  under  favorable  con- 
ditions resist  the  same  solution  for  several  days.  From  what  has  already  been  said, 
however,  it  must  be  clear  that  such  statements  are  true  only  in  variable  degree,  accord- 
ing to  the  medium  in  which  the  organisms  to  be  destroyed  may  be  contained.  A  much 
shorter  period  of  exposure  and  a  greater  dilution  of  the  disinfectant  will  accomplish 
destruction  of  bacteria  suspended  in  an  albumin-free  liquid  than  could  be  brought  about 
in  the  same  time  by  even  stronger  solutions  of  the  chemical  acting  upon  the  same  organ- 
isms embedded  in  a  highly  albuminous  mass  or  in  a  strongly  albuminous  liquid.  In 
determining  the  germicidal  value  (or  the  antiseptic  value  as  well)  of  any  substance,  there- 
fore, for  the  sake  of  uniformity  the  germs  exposed  to  disinfection  should  invariably  be 
suspended  in  the  same  type  of  surrounding  medium,  a  one  per  cent,  peptone  solution 
being  commonly,  selected  for  the  purpose. 

For  the  determination  of  the  disinfectant  value  of  any  substance  there  are  a 
number  of  methods  in  vogue,  one  of  the  most  simple  and  satisfactory  of  wrhich  is  that 
devised  by  Sternberg,  the  principles  of  which  are  followed  in  these  directions:  Several 
tubes,  each  containing  a  known  quantity  of  virulent  culture  of  a  given  organism  in 
peptone  solution,  are  prepared.  To  each  a  definite  amount  of  the  disinfectant  to 
be  tested  is  added,  so  as  to  make  a  series  of  dilutions  of  the  germicide  in  known  propor- 
tions. Each  is  agitated  so  as  to  thoroughly  diffuse  the  disinfectant,  and  at  regular  in- 
tervals inoculations  are  made  from  each  into  fresh  tubes  of  the  nutrient  material, 
incubated,  and  the  results  noted  as  usual  at  the  close  of  each  day  for  three  days. 
That  tube  representing  the  least  proportion  of  the  disinfectant,  acting  for  the  shortest 
period,  in  ivhich  no  growth  follows,  corresponds  to  the  disinfectant  value  of  the  substance  for 
the  selected  microorganism.  If  it  be  objected  that  the  small  amount  of  the  disinfectant 
transferred  in  the  inoculation  of  the  fresh  tubes  has  exercised  in  these  tubes  a  restrictive 
influence,  this  may  be  estimated  and  corrected  by  a  control  experiment,  by  taking  the 
same  amount  (a  loopful)  of  the  disinfectant  substance  in  the  same  degree  of  dilution  in 
peptone  solution  as  that  represented  by  the  original  tube  (that  corresponding  to  the 
accepted  germicidal  value  of  the  disinfectant),  transferring  it  to  a  second  tube  of  the 


48  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

same  medium,  diffusing  it  by  gentle  agitation,  and  then  planting  therein  from  a  virulent 
culture  of  the  germ  employed  in  the  test,  so  as  to  note  whether  such  proportion  exercises 
any  destructive  or  restraining  influence. 

Exercise  16. — Prepare  four  tubes  of  one  per  cent,  peptone  solution, 
each  containing  four  cubic  centimeters;  inoculate  each  with  the  colon 
bacillus  and  incubate  for  twenty-four  hours,  protecting  the  contents  of 
the  tubes  against  evaporation  by  a  rubber  cap  or  rubber  stopper  over  the 
usual  cotton  plug.  Having  the  following  day  made  certain  of  the  success 
of  the  infection  of  each  tube,  add  to  the  first  one  cubic  centimeter  of  a 
five  per  cent,  solution  of  carbolic  acid  (thus  making  a  one  per  cent,  dilu- 
tion of  the  acid  in  this  tube) ;  to  the  second,  one  cubic  centimeter  of  a  ten 
per  cent,  solution  (two  per  cent,  solution  in  the  tube) ;  to  the  third,  one 
cubic  centimeter  of  a  fifteen  per  cent,  solution  (a  three  per  cent,  solution 
in  tube);  and  to  the  fourth,  one  cubic  centimeter  of  a  twenty  per  cent, 
solution  (four  per  cent,  in  the  tube).  Diffuse  the  disinfectant  immediately 
in  each  by  gentle  agitation.  After  appropriate  intervals  (five,  ten,  twenty, 
thirty,  forty,  fifty,  and  sixty  minutes)  transfer  with  the  platinum  loop 
one  loopful  from  each  tube  to  a  fresh  tube  of  the  peptone  solution.  Each 
tube,  properly  marked,  is  incubated,  and,  at  the  usual  intervals  of  one, 
two,  and  three  days,  observations  are  made  and  recorded.  What  tubes 
permit  growth?  What  tubes  show  no  development?  That  tube  planted 
from  the  greatest  dilution  of  the  carbolic  acid,  having  had  the  shortest 
action  period,  which  shows  no  growth,  may  be  taken  as  representing  the 
disinfectant  value  of  this  substance  for  the  colon  bacillus.  To  verify 
the  result,  make  a  preparation  of  the  same  degree  of  dilution  of  the  acid 
in  a  sterile  tube  of  the  peptone  solution,  and  inoculate  it  with  a  loopful 
of  a  virulent  culture  of  the  organism.  If  growth  readily  follows,  it  may 
be  inferred  that  no  material  influence  was  exercised  by  the  small  amount 
of  carbolic  acid  transferred  in  the  test  experiment;  if  growth  occurs  but 
slowly,  retardation  only  may  have  been  accomplished,  and  in  such  event 
the  contents  of  the  tube  accepted  as  indicating  the  disinfecting  power 
of  the  acid  in  the  test  experiment  should  be  diluted  freely  with  sterile 
peptone  solution,  further  incubated,  and  observed.  If  no  growth  should 
follow,  however,  it  may  be  accepted  for  practical  purposes  that  actual 
disinfection,  and  not  mere  retardation  (antisepsis),  was  accomplished. 

Determination  of  Antiseptic  Values. — It  should  be  recalled  here,  as  in  the  case 
of  disinfection,  that  the  antiseptic  value  must  vary  for  every  substance  according  to  the 
microorganism  subjected  to  its  influence,  and  probably  according  to  the  character  of  the 
surrounding  medium  as  well.  The  method  for  determination  is  a  simple  one,  consisting  of 
the  addition  of  known  amounts  of  the  material  under  investigation  to  known  amounts 
of  sterile  nutrient  medium  (one  per  cent,  peptone  solution),  each  tube  thus  prepared 


50  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

being  then  inoculated  with  the  test  bacteria.  That  tube  containing  the  least  amount  of 
the  antiseptic  in  which  growth  fails  to  appear  represents  the  antiseptic  value  of  that 
substance  in  relation  to  the  particular  bacterium  used  in  the  test.  However,  in  such 
tube  it  should  be  possible,  upon  the  addition  of  an  excess  of  the  peptone  solution  (thus 
further  diluting  the  antiseptic),  to  obtain  the  characteristic  development  of  the  organ- 
ism. 

Exercise  17. — Four  tubes,  each  containing  four  cubic  centimeters  of 
a  one  per  cent,  solution  of  peptone,  are  prepared.  To  the  first  is  added 
one  cubic  centimeter  of  a  two  per  cent,  solution  of  borax  (making  a  i :  250 
solution  of  the  borax  in  the  tube] ;  to  the  second,  one  cubic  centimeter  of  a 
one  per  cent,  solution  of  borax  (i :  500  solution  in  tube} ;  to  the  third,  one 
cubic  centimeter  of  a  half  per  cent,  solution  of  the  borax  (i :  1000  solu- 
tion in  tube) ;  to  the  fourth,  one  cubic  centimeter  of  a  quarter  per  cent, 
solution  of  the  borax  (i  12000  solution  in  the  tube).  Inoculate  each  tube 
with  a  virulent  culture  of  the  colon  bacillus,  and  incubate  for  three  days. 
As  a  control,  a  tube  of  the  same  medium  is  to  be  inoculated  at  the  same 
time  with  the  germ,  no  borax  having  been  introduced.  Note  the  ap- 
pearances presented  by  each  tube  at  the  close  of  each  twenty-four  hours. 
At  the  end  of  the  third  day  select  the  tube  which  fails  to  show  any  bac- 
terial contamination,  haying  the  least  proportion  of  borax  present.  Di- 
lute the  contents  with  an  equal  amount  of  sterile  peptone  solution  and 
again  incubate.  Growth  should  now  follow.  Should  it  fail  it  is  to  be 
presumed  that  the  proportion  of  borax  contained  therein  has  acted  rather 
as  a  disinfectant;  and  the  tube  containing  the  next  lower  proportion  of 
the  borax  should  be  similarly  treated  to  determine  the  persistence  of 
vitality  of  the  bacilli.  The  first  tube  of  the  series  which  thus  presents 
growth  after  dilution  with  more  of  the  culture  medium,  but  which  before 
dilution  failed  to  exhibit  development  of  the  organisms  which  had  been 
transplanted  therein,  represents  the  antiseptic  value  of  borax  for  the 
colon  bacillus. 

Exercise  18. — To  demonstrate  the  possible  failure  of  a  disinfectant 
because  of  the  formation  of  a  protective  layer  of  organic  compounds  of 
the  disinfectant  with  the  nutrient  medium  or  with  the  outer  portion  of 
the  bacterial  wall,  thus  encasing  the  bacteria  or  their  spores  and  pre- 
venting proper  contact  between  the  disinfectant  and  the  microorganismal 
body,  the  following  exercise  should  be  practised :  Suspend  for  several 
hours  five  bits  of  sterilized  silk  or  cotton  thread,  each  about  two  inches 
in  length,  in  a  bouillon  culture  of  the  hay  bacillus  (B.  subtilis),  which 
has  been  grown  in  the  incubator  for  three  or  four  days.  The  threads 
after  removal  are  suspended  in  an  empty  sterile  tube  and  dried  at  incu- 
bator temperature.  They  are  next  suspended  in  a  i :  1000  solution  of 


52  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

mercuric  chloride.  After  five  minutes  one  of  the  threads  is  withdrawn 
with  a  sterile  (flamed)  forceps,  and  divided  into  equal  lengths  by  means 
of  a  sterilized  scissors.  One  of  the  latter  bits  is  at  once  transferred  to 
a  fresh  tube  of  sterile  nutrient  substance.  The  second  is  gently  washed 
in  sterile  water  to  free  it  from  the  excess  of  the  sublimate  solution;  then 
washed  for  half  a  minute  in  a  weak  (two  per  cent.)  solution  of  ammonium 
sulphide  in  order  to  remove  any  possible  coat  of  mercurial  albuminate 
from  about  the  microorganisms  and  their  spores.  This  is  then  rinsed  off 
by  second  immersion  of  the  thread  in  sterile  water,  after  which  the  thread 
is  transferred  to  a  tube  of  sterile  nutrient  medium.  The  same  procedures 
are  performed  with  the  remaining  bits  of  thread  at  the  close  of  ten  minutes, 
twenty  minutes,  and  thirty  minutes.  Each  tube,  properly  marked,  is 
then  placed  in  the  incubator  and  observations  made  at  the  close  of  the 
first,  second,  and  third  days. 

Exercise  19. — Practise  one  of  the  methods  of  surgical  disinfection  of 
the  hands,  inoculating  sterile  bouillon  with  scrapings  from  the  hands  and 
material  beneath  the  nails  before  the  process  as  controls,  and  similarly 
planting  scrapings  from  the  same  sources  after  each  stage  of  the  pro- 
cedure in  order  to  determine  the  value  of  the  several  steps  of  the  process. 
In  this  exercise  let  the  class  be  arranged  in  groups  of  five,  each  individual 
planting  the  preliminary  control  tubes  from  the  hand  and  nails.  In 
each  group  let  one  man  carry  out  the  process  in  full  before  making  the 
second  inoculation.  Let  the  second  wash  the  hands  with  soap  and  hot 
water,  using  a  sterile  hand  brush  for  effectiveness;  when,  having  rinsed 
the  hands  in  sterile  water,  let  him  make  inoculations  from  the  epiderm 
and  nails  into  sterile  bouillon.  The  third  should  soak  his  hands  for  five 
minutes  in  a  i :  1000  solution  of  bichloride  of  mercury,  working  the  dis- 
infectant well  into  the  skin  by  means  of  a  sterilized  hand  brush; 
after  which,  having  rinsed  his  hands  free  from  the  disinfectant  in 
sterile  water,  let  him  make  similar  inoculations  from  the  epiderm  and 
nail  scrapings  into  sterile  bouillon.  A  fourth  should  soak  his  hands  in 
a  saturated  solution  of  potassium  permanganate,  rinse  off  the  excess  in 
sterile  water,  and  make  similar  inoculations.  The  fifth  should  soak  his 
hands  in  a  solution  (saturated)  of  oxalic  acid,  rinse  the  hands  in  sterile 
water,  and  likewise  inoculate  tubes  of  sterile  bouillon  with  scrapings  from 
the  skin  surface  and  from  the  nails.  Further,  the  third  man  of  each 
group,  who  has  sterilized  his  hands  with  the  sublimate  solution  alone, 
should  now  soak  his  hands  in  a  two  per  cent,  solution  of  ammonium  sul- 
phide, brushing  it  well  in,  and  again  rinse  off  the  chemical  and  make 
inoculations  from  the  same  sources  as  before.  Moreover,  the  first  man, 
who  has  carried  out  the  entire  process  of  hand  sterilization,  should  make 


54  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

deep  scrapings  from  the  epiderm,  inoculating  them  as  before,  in  order  to 
determine  the  degree  of  penetration  of  the  disinfectant  solutions  below 
the  surface  of  the  skin.  All  tubes,  properly  marked,  are  now  placed  into 
the  incubator;  and  at  the  usual  intervals  of  one,  two,  and  three  days 
observations  made  in  comparison  with  the  control  experiments  and  the 
results  noted. 

Disinfectant  Gases. — Disinfection  of  large  spaces,  as  of  sick-rooms,  hospital  wards, 
ship-holds,  and  similar  inclosures,  is  commonly  attempted  by  means  of  disinfectant 
gases ;  the  process  being  completed  by  washing  the  floors,  walls,  and  ceilings  as  far  as 
practicable,  and  the  furniture,  when  possible,  with  disinfectant  solutions,  as  of  corrosive 
sublimate  or  carbolic  acid,  all  comparatively  worthless  material  burned,  and  all  material 
which  can  be  safely  and  conveniently  subjected  to  heat  thoroughly  baked,  boiled,  or 
steamed  in  the  usual  manner.  The  gases  commonly  used  for  such  purpose  are  formalde- 
hyde and  sulphurous  oxide.  When  the  method  is  attempted  it  is  essential  for  success 
that  the  exposure  shall  be  a  prolonged  one,  the  room  being  kept  filled  with  the  gas  for 
twenty-four  hours,  and  all  cracks  and  crevices  about  doors  and  windows  carefully  closed 
with  strips  of  paper  pasted  over  them,  or  by  cotton  or  rag  caulking,  to  prevent  the  escape 
of  the  gas.  Of  course,  all  large  openings,  as  of  flues,  registers,  ventilating  openings,  as 
well  as  doors  and  windows,  are  to  be  thoroughly  sealed.  All  clothes,  curtains,  carpets, 
and  similar  substances  which  it  is  desired  to  have  disinfected  by  the  gas  are  to  be  hung 
about  the  apartment  in  the  thinnest  possible  layers  so  as  to  facilitate  thorough  penetra- 
tion. When  sulphur  is  to  be  used,  it  is  best  that  water  should  first  be  evaporated  in  the 
room  so  as  to  make  the  atmosphere  as  moist  as  possible,  such  moisture  aiding  in  the 
penetration  of  the  gas  into  any  fabric  exposed ;  it  is  not  essential,  however,  in  case  for- 
maldehyde is  employed,  but  is  not  objectionable.  In  practice  it  is  customary  to  burn 
twenty  grams  of  sulphur  for  each  cubic  meter  of  space,  the  sulphur  being  either  in  the 
form  of  the  sulphur  disinfecting  candles  or  in  the  powder,  when  it  is  well  to  first  moisten 
it  with  alcohol.  Care  should  be  taken  to  protect  the  floor  and  the  room  contents  from 
the  sulphur  flame,  either  by  burning  the  sulphur  in  candle  form  floating  upon  a  wide 
dish  of  water  (the  sulphur  upon  a  board  or  in  a  shallow  dish),  or  burning  the  powdered 
sulphur  in  a  dish  set  in  a  large  sand  or  earth  bath. 

In  using  formaldehyde,  from  ten  to  fifteen  cubic  centimeters  of  the  commercial 
solution  should  be  evaporated  for  each  cubic  meter  of  space.  A  number  of  forms  of 
apparatus  have  been  devised  for  the  purpose  of  generating  this  gas ;  of  these,  probably 
the  most  efficient  are  those  in  which  the  commercial  formaldehyde  solution,  mixed  with 
glycerine  or  calcium  chloride  to  prevent  its  conversion  into  its  solid  isomer  paraform,  is 
vaporized  in  a  metal  retort  or  tube,  by  means  of  an  alcohol  flame  beneath,  the  gas  being 
discharged  from  the  mouth  of  the  apparatus  through  a  tube,  by  which  it  may  be  con- 
veyed through  a  keyhole  or  other  small  aperture  into  the  interior  of  the  room  to  be  dis- 
infected. Or  the  gas  may  be  obtained  by  heating  the  paraform,  which  may  be  obtained 
in  pastile  form  in  commerce ;  or  it  may  be  produced  by  the  action  of  a  heated  plate  of 
copper  upon  the  fumes  of  wood  alcohol.  Special  types  of  generators  based  upon  each 
of  the  latter  methods  may  be  had  and  will  serve  the  required  purpose. 

Exercise  20. — Three  slips  of  sterile  filter  paper  or  thread  are  soaked 
in  an  active  culture  of  one  of  the  pus  germs,  as  of  the  Micrococcus  pyogenes 
aureus,  for  an  hour.  They  are  then  separately  inclosed,  each  in  a  definite 


56  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

but  varying  number  of  layers  of  blanket  cloth,  and  distributed  to  the 
top,  floor,  and  middle  height  of  a  room  of  known  dimensions,  which  has 
previously  been  prepared  as  above  outlined  for  the  prevention  of  the 
escape  of  the  disinfectant  gas  used.  Formaldehyde  is  generated  by  any 
convenient  form  of  generator  and  passed  in  the  above-mentioned  pro- 
portion into  the  interior  of  the  room,  which  is  thereafter  kept  closely 
sealed  for  twenty-four  hours,  then  opened  and  well  aired.  As  soon  as 
convenient  the  room  is  entered  and  the  packages  containing  the  infected 
slips  of  paper  removed  and  inoculations  made  therefrom  into  sterile 
bouillon,  the  tubes  marked,  placed  in  the  incubator,  and  observations 
made  after  the  first,  second,  and  third  days  and  the  results  recorded. 

Exercise  21. — The  above  experiment  is  to  be  repeated,  substituting 
sulphurous  oxide  for  the  formaldehyde  gas, 

Risumi. — From  the  foregoing  exercises  it  should  be  possible  to  come  to  fair  con- 
clusions as  to  the  relative  merits  of  the  various  methods  of  sterilization  suggested ;  and 
it  is  expected,  therefore,  that  the  student  will  in  review  calculate  the  actual  number  and 
percentage  proportion  of  successful  sterilizations  accomplished  by  the  class  in  the  vari- 
ous full  applications  of  heat,  and  by  nitration  and  chemical  disinfection. 


LESSON  HI. 

PREPARATION  OF  TUBES,  FLASKS,  DISHES,  ETC. 

In  the  study  of  bacteria  the  first  object  before  the  investigator  is  to  obtain,  isolated 
from  all  other  forms  of  microorganisms,  a  sufficient  growth  of  the  particular  type  to 
afford  opportunity  for  observation  of  its  characteristics.  Such  an  isolated  growth  is 
spoken  of  as  a  pure  culture;  where  several  varieties  are  more  or  less  mingled  in  their 
development,  the  term  mixed  or  impure  culture  is  used.  The  artificial  cultivation  of 
these  organisms  is  accomplished  by  affording  them  conditions  for  growth  more  or  less 
similar  to  those  required  by  them  in  nature  (nutrient  material,  a  definite  range  of  tem- 
perature, moisture,  and  proper  atmosphere).  For  the  purpose  of  preserving  the  nu- 
trient material  free  from  other  organisms  than  those  purposely  implanted  upon  it,  and 
to  aid  in  maintaining  perfect  isolation  of  the  various  cultures,  there  is  to  be  provided 
no  little  apparatus,  the  principal  forms  of  which  may  here  be  briefly  considered. 

i.  Test-tubes.— A  large  number  of  test-tubes,  the  most  commonly  employed  form 
of  container  for  culture  media,  should  always  be  available.  The  ordinary  chemical  test- 
tubes  may  be  used  for  this  purpose,  but  it  is  preferable  that  the  tubes  should  be  some- 
what heavier  and  made  of  a  glass  which  will  not  be  corroded  by  the  process  of  steriliza- 


FIG.   ii. — TEST-TUBE  BRUSH. 

tion.  They  may  be  plain,  or  provided  with  the  usual  flange  about  the  mouth,  as  suits 
the  fancy  of  the  individual.  A  convenient  size  is  five  inches  in  length  and  five-eighths  of 
an  inch  diameter;  and  it  will  be  found  advantageous  to  have  a  small  portion  of  the 
outer  surface  of  the  upper  part  of  each  tube  roughened  by  etching  with  "white  acid," 
for  the  purpose  of  receiving  pencil  marks  in  labeling.  A  larger  size  is  usually  provided 
for  the  reception  of  potato  cylinders  sometimes  employed  as  culture  media.  Moreover, 
it  is  well  to  have  a  few  very  large  tubes  on  hand  for  use  in  making  anaerobic  cultures  and 
as  sedimentation  tubes  in  the  preparation  of  solidified  blood-serum. 

Cleansing  New  Tubes. — Before  the  nutrient  media  are  introduced  into  the  tubes  the 
latter  are  to  be  carefully  cleansed,  the  mouth  of  each  plugged  with  a  cotton  stopper,  and 
the  whole  sterilized.  In  preparing  tubes  which  have  not  been  previously  used,  ordinary 
washing  in  water  with  a  test-tube  brush  (Fig.  1 1)  followed  by  soaking  for  from  five  to  ten 
minutes  in  a  weak  solution  of  one  of  the  mineral  acids  (as  a  one  per  cent,  solution  of 
hydrochloric  acid)  to  neutralize  any  of  the  remaining  alkali  employed  in  manufacture, 
and  by  rinsing  in  clean  water,  will  be  found  sufficient.  The  tubes  are  then  to  be  in- 
verted on  the  draining  board  to  dry. 

Cleansing  Old  Tubes, — When  tubes  have  been  previously  used  for  culture  purposes 

58 


60 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


and  perhaps  are  contaminated  by  infectious  material,  the  process  of  cleansing  requires 
more  care  and  detail.  It  is  not  free  from  possible  danger,  and  the  student  should  exer- 
cise all  the  caution  and  fulfil  all  the  steps  indicated  in  the  folio  wing  directions  for  its  per- 
formance. The  contaminated  tubes  having  been  collected,  the  operator  should  remove 
the  old  cotton  stoppers,  grasping  them  with  a  strong  pair  of  dissecting  forceps  (subse- 
quently to  be  sterilized  by  flaming),  and  deposit  them  at 
once  in  one  of  the  laboratory  waste  jars  containing  a  large 
amount  of  one  of  the  disinfectant  solutions.  This  material 
should  be  burned  subsequently.  Each  tube,  after  the  re- 
moval of  the  stopper,  is  to  be  filled  even  with  its  lip  with 
a  strong  disinfectant  solution  (as  water  containing  five  per 
cent,  each  of  carbolic  and  hydrochloric  acids),  and  allowed 
to  stand  for  from  twelve  to  twenty-four  hours,  in  which  in- 
terval it  is  probable  the  destruction  of  any  living  infection 
will  have  been  accomplished.  The  contents  of  the  tubes 
are  now  poured  with  as  little  spattering  as  possible  into 
one  of  the  waste  jars  and  subsequently  burned.  As  emp- 
tied, the  tubes  are  placed  in  a  suitable  vessel  containing 
water  to  which  has  been  added  sufficient  washing  soda  to 
render  it  distinctly  alkaline  (two  or  three  per  cent.) ,  in  which 

they  are  to  be  boiled  for  an  hour.  This  done,  there  is  no  longer  danger  of  infection,  and 
each  tube  is  now  thoroughly  washed  and  brushed  in  clean  hot  water,  the  outside  surface 
being  wiped  clean  with  a  wash-cloth.  The  tubes  are  next  placed  for  a  few  minutes  in 
a  weak  solution  of  one  of  the  mineral  acids,  as  in  case  of  new  tubes,  to  neutralize  any 
remaining  alkali,  rinsed  in  clean  water  and  placed  in  inverted  position  on  the  draining 
board  to  dry. 


FIG.    12. — WIRE   BASKET 
FOR  TEST-TUBES. 


'*0r 

FIG.   13. — Roux's   POTATO 
TUBE. 


FIG.  14. — FERMENTATION 
TUBE. 


—vmf— 

YIG.  15. — FERMENTATION 
TUBE. 


Cotton  Plugs. — The  next  step  of  the  preparation  is  the  application,  in  the  open  end 
of  each  tube,  of  a  cotton  stopper  to  prevent  the  entrance  of  contaminating  organisms 
from  without  (v.  Ex.  13).  These  stoppers  should  be  of  such  a  size  that  when  fitted  into 
the  mouth  of  the  tube  the  latter  may  be  sustained  easily  when  lifted  by  the  hand  grasp- 


62 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


ing  the  protruding  end  of  the  stopper;  however,  the  cotton  should  not  be  too  closely 
packed  into  the  tube,  and  should  have  been  rolled  into  an  even  cylinder  rather  than 
loosely  and  unevenly  crowded  into  the  tube.  Ordinarily  common  cotton  batting  is 
used  for  the  purpose ;  the  only  objection,  however,  to  absorbent  cotton  is  its  cost.  Bat- 
ting will  assume,  with  proper  sterilizing  heat  to  which  the  tubes  are  subsequently  ex- 
posed, a  light  brown  hue  which  may  be  taken  as  a  rough  indicator  of  proper  sterilization ; 


FIG.  1 6. — TYPES  OF  CULTURE  FLASKS. 

however,  if  higher  temperatures  should  accidentally  be  attained,  batting  will  present  a 
disadvantage  in  that  a  dark  oil  is  driven  from  the  cotton,  which  will  enter  the  tubes  as  a 
vapor  and  condense  upon  the  inner  surface.  If  permitted  to  remain  it  is  apt  to  interfere 
with  the  development  of  inoculations,  is  unsightly,  and  should  be  removed  by  thorough 
washing. 

Sterilization  of  Tubes. — After  the  tubes  have  been  cleaned  and  the  cotton  stoppers 

adjusted,  they  are  placed  in  a  basket 
made  of  wire  netting  (Fig.  12),  and  steril- 
ized by  baking  in  the  dry-air  oven  for  an 
hour  at  a  temperature  of  140°  to  150°  C. 
(v.  Ex.  7  and  8).  It  is  not  essential  that 
the  complete  fractional  heating  should  be 
carried  out  in  this  preliminary  process  (ex- 
cept in  special  cases,  as  when  the  tubes 
are  intended  for  blood-serum),  since  after 
the  introduction  of  the  media  the  thorough 
sterilization  of  the  latter  as  well  as  of  the 
tube  will  be  necessary. 

Special  Forms. — In  preparing  tubes  for  potatoes,  if  the  tubes  be  of  the  common 
form,  it  is  well,  before  adjustment  of  the  cotton  stoppers,  to  place  in  the  interior  of  each 
a  small  bit  of  glass  tube  or  rod  or  a  wad  of  cotton  to  serve  as  a  support  for  the  potato 
cylinder  in  order  to  prevent  the  latter  from  descending  to  the  bottom  of  the  tube,  where 
in  the  process  of  sterilization  there  will  collect  more  or  less  liquid.  This  liquid  is  re- 
tained in  the  tube  to  afford  moisture  to  colonies  of  bacteria  subsequently  grown  upon 
the  surface  of  the  potato.  A  special  form  of  tube  is  sometimes  used  to  obviate  the  need 


FIG.  17. — TYPES  OF  DISTRIBUTION  FLASKS. 


64 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


for  such  rest,  as  shown  in  Fig.  13  ;  however,  there  is  added 'difficulty  in  cleaning  this  tube, 
and  it  is  therefore  of  questionable  advantage. 

In  the  study  of  gas  formation  by  bacteria,  fermentation  tubes  of  the  same  form  as 
those  used  in  urinalysis  for  sugar  fermentation  and  for  the  estimation  of  urea  (Doremus' 
ureometer)  are  usually  used  (Fig.  14).  The  collection  arm  of  such  tubes  need  not  be 
graduated,  but  the  total  capacity  of  the  arm  should  be  ascertained  so  that  the  amount 
of  gas  may  be  approximately  calculated  in  special  cases.  An  efficient  substitute  for 
such  fermentation  tubes  may  be  extemporized  by  inverting  a  small  test-tube  filled  with 
the  medium  in  a  larger  one,  after  the  principle  of  a  gas  jar  in  a  water-bath  (Fig.  15). 


FIG.  18.— PETRI  DISH. 

2.  Flasks. — It  is  customary  to  employ  flasks  as  containers  for  the  nutrient  media 
in  bulk,  distribution  to  culture  tubes  being  made  from  time  to  time  as  necessity  de- 
mands. It  is  best  for  this  purpose  to  use  the  small  sizes  (as  those  of  250  to  500  cubic 
centimeters  capacity),  since,  should  accidental  contamination  occur,  smaller  quantities 
of  the  media  are  endangered.  When  large  amounts  of  a  culture  of  some  special  organ- 
ism are  desired,  flasks  are  often  used  instead  of  tubes  or  dishes,  and  a  number  of  special 
forms  of  culture  flasks  may  be  obtained  from  the  makers  (Fig.  16).  So,  too,  a  number 
of  special  forms  of  flasks  have  been  devised  for  the  storage  of  media  and  their  ready  and 


FIG.  19. — PETRI  DISH. 


safe  distribution  to  tubes  (Fig.  17).  For  general  use,  however,  the  ordinary  flat-bot- 
tomed Erlenmeyer  flask  is  deservedly  the  most  popular,  its  shape  being  favorable  in 
cleaning  and  its  broad,  flat  bottom  giving  it  stability. 

Flasks  are  to  be  cleaned,  stoppered,  and  sterilized  before  use  in  precisely  the  same 
manner  as  detailed  for  the  culture  tubes.  Sand  or  shot  agitated  in  the  water  will  facili- 
tate the  cleansing  of  the  interior,  but  it  is  best  to  use  a  long-handled  brush  obtainable 
for  the  purpose. 

3.  Dishes. — Two  types  of  dishes  are  in  general  use — the  large  form  used  for  potato 
and  plate  cultures,  and  the  small  Petri  dishes. 


66 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


(a)  Potato  Dishes. — These  are  large  flat  dishes,  usually  about  twenty  centimeters  in 
diameter  and  about  eight  centimeters  in  height,  with  straight  sides,  and  fitted  with  covers 
of  the  same  shape  as  the  dish  itself,  but  of  slightly  greater  diameter,  so  as  to  permit  the 
cover  to  easily  fit  down  over  the  dish  (Fig.  18).  They  are  used  to  protect  the  large  slices 
of  potatoes  often  used  as  culture  material  or  the  sets  of  plates  in  plate  cultures.  Be- 
cause of  their  size  and  the  quality  and  texture 
of  glass  used  in  their  manufacture  they  cannot 
well  be  subjected  to  heat  in  sterilization. 
Therefore,  after  having  been  well  washed  and 
cleansed  after  the  method  detailed  for  test- 
tubes  (omitting  the  boiling),  it  is  customary 
to  place  in  the  bottom  of  each  dish  a  layer  of 
ordinary  filter  paper  (intended  to  retain 
moisture  for  cultures),  after  which  the  dish, 
and,  too,  the  cover,  are  filled  level  full  with 
a  disinfectant  solution  (1:1000  solution  of 
mercuric  chloride) ,  which  is  allowed  to  remain 
therein  for  at  least  one  hour  (in  practice  often 
over  night).  The  sublimate  solution  is  then 
poured  out  and  the  cover  at  once  adjusted, 
after  which  the  potatoes  or  plates  may  be 
introduced.  There  are  numerous  opportuni- 
ties, in  the  adjustment  of  the  cover  and  at 
tj.me  of  introduction  of  potatoes  and  plates, 
for  the  contamination  of  the  interior  by  or- 
ganisms from  the  air,  and  the  protection  of 
the  media  by  such  dishes  is  therefore  not  to 

be  compared  with  that  obtained  in  tubes  or  flasks ;  their  use,  therefore,  is  by  no  means 
so  frequent  as  formerly.  Care  should  be  exercised,  in  the  selection  of  the  dishes  and 
covers,  that  the  contact  between  the  cover  and  the  edge  of  the  dish  be  as  perfect  as 
possible.  * 

(6)  Petri  Dishes. — These  dishes  (Fig.  19),  of  the  same  general  appearance  as  the 
large  culture  dishes,  but  much  flatter,  are  usually 
eight  or  ten  centimeters  in  diameter  and  one 
and  a  half  to  two  centimeters  in  height.  They 
were  introduced  to  take  the  place  of  the  plates 
employed  in  the  separation  of  bacteria  in  impure 
cultures,  and  are  of  particular  service,  as  they 
permit  the  examination  of  the  gross  colonies  of 
organisms  developing  in  the  interior  by  means 
of  the  microscope,  the  unopened  dish  being 
placed  upon  the  stage  of  the  instrument  and 
examined  with  the  low  powers  of  the  microscope. 
The  same  precautions  in  the  cleansing  of  these 

dishes  are  to  be  observed  as  detailed  for  tubes.  The  covers  are  then  applied  to  the 
dishes  and  the  whole  sterilized  in  the  dry-air  oven  by  the  fractional  method  (the  whole 
process  should  be  performed,  as  there  is  usually  no  opportunity  for  further  steriliza- 
tion after  the  introduction  of  the  culture  medium).  It  will  be  found  of  advantage 
to  fold  the  dish  and  cover  in  wrapping  paper  before  sterilization,  the  paper  being  allowed 


FIG.  20. — METAL  Box  FOR  HOLDING 
GLASS  PLATES  DURING  STERILIZA- 
TION IN  OVEN. 


FIG. 


21. — SET  OF  CULTURE  PLATES 
AND  PLATFORMS. 


68  LABORATORY  EXERCISES  IN  BACTERIOLOGY, 

to  remain  until  they  are  to  be  used,  in  order  to  prevent  accidental  separation  of  the 
covers  from  the  dishes.  Contamination  of  the  interior  by  bacteria  from  the  surround- 
ing atmosphere  is,  as  in  case  of  the  potato  dishes  (but  to  a  less  degree  because  of  the 
smaller  size  of  the  Petri  dishes  and  the  better  adaptation  of  their  covers),  more  likely  to 
take  place  than  in  the  use  of  culture  tubes ;  but  this  can  in  some  measure  be  obviated 
by  the  application  of  a  strip  of  paper  or  a  rubber  band,  such  as  is  used  about  bank  books, 
about  the  outside  surface  along  the  line  of  overlapping  of  the  cover. 

4.  Plates. — Plates  of  glass,  usually  four  by  five  inches  in  size,  were  formerly  much 
used  in  the  separation  of  the  bacteria  of  impure  cultures  to  obtain  pure  cultures,  the 
inoculated  medium  being  spread  over  the  surface  of  such  a  plate  so  that  the  organisms 
present  might  in  development  grow  into  well-separated  colonies  and  thus  favor  the  re- 
moval of  such  as  may  be  desired,  by  means  of  the  needle,  to  fresh  culture  tubes.  They 
are  occasionally  used  at  present  for  this  purpose,  but  have  been  largely  superseded  by 
Petri  dishes  and  by  the  so-called  Esmarch  tubes.  In  preparation  for  use,  the  plates  are 
cleaned  in  the  same  manner  as  indicated  for  tubes  and  are  then  dried  and  placed  in  a 
metal  box  with  close-fitting  lid,  in  which  they  are  baked  in  the  dry-air  oven  in  the  usual 
manner  of  fractional  sterilization.  The  box  is  opened  only  when  the  plates  are  required 
to  be  withdrawn  for  use  (Fig.  20).  In  arranging  plate  cultures  it  is  customary  to  place 
these  plates  in  the  large  culture  dishes  above  mentioned,  usually  three  in  each  dish,  each 
supported  upon  a  glass  platform  and  built  into  a  "set,"  one  over  the  other  (Fig.  21). 
The  glass  platforms  are  to  be  prepared  for  use  in  the  same  way  as  the  plates,  but  when 
sterilized  should  be  wrapped  separately  in  paper,  which  is  kept  about  them  for  protec- 
tion until  they  are  to  be  arranged  in  the  culture. 

In  addition  to  the  above  apparatus  there  will  be  required  a  number  of  other  forms 
of  glassware  and  other  appliances  for  the  preparation  and  distribution  of  the  culture 
media  and  for  the  prosecution  of  culture  experiments,  which  may  be  described  to  best 
advantage  in  connection  with  the  processes  in  which  they  are  employed. 

Exercise  22. — Let  the  student  at  this  time  prepare,  according  to  the 
preceding  instruction,  half  a  gross  of  ordinary  culture  tubes,  one  dozen 
potato  tubes,  ten  Petri  dishes,  and  clean  all  tubes,  etc.,  which  have  been 
used  in  previous  exercises. 


LESSON   IV. 

CULTURE  MEDIA. 

A  variety  of  nutrient  substances  are  in  more  or  less  general  use  in  the  culture  of 
bacteria,  and  such  variety  is  essential,  not  merely  for  the  ordinary  requirement  of 
growing  the  organisms,  but  also  for  the  further  purpose  of  observation  of  the  varying 
characteristics  presented  by  this  or  that  bacterium  upon  the  different  types  of  media 
and  furnishing  data  useful  for  its  identification.  Of  the  different  media  suggested, 
the  most  important  for  routine  use  are  potatoes,  bouillon,  gelatinized  bouillon  ("gelatine"}, 
agarized  bouillon  ("agar"),  peptone  solution,  glucose-,  lactose-,  and  saccharose-bouillon, 
blood-serum,  milk,  and  litmus-milk.  In  the  preparation  of  these  media  it  is  important, 
inasmuch  as  from  variation  of  their  constitution  there  may  follow  important  alterations 
of  the  characteristics  of  the  bacteria  grown  upon  them,  that  uniformity  of  manufacture 
should  be  sought  in  order  that  the  records  of  different  individuals  engaged  upon  the 
same  microorganisms  may  be  uniform  and  comparable.  It  is  desirable,  too,  that  all 
results  observed  as  to  the  growth  of  any  bacterium  upon  the  standard  or  upon  special 
modifications  of  the  common  media  should  be  recorded  with  clear  indication  of  the  exact 
composition  of  the  nutrient  medium  employed,  as  well  as  the  other  conditions  of  growth 
prevailing.  The  reaction  of  the  medium  is  an  especially  important  feature;  in  which 
matter  it  is  to  be  recommended  that  the  standard  reaction  adopted  by  the  Bacterio- 
logic  Committee  of  the  American  Public  Health  Association  be  followed.  It  is  to 
be  understood,  except  as  may  be  indicated  in  the  following  instructions,  that  the  reaction 
of  all  media  described  should  be  adjusted  to  this  standard:  that  to  phenolphthalein  (a 
more  delicate  reaction  indicator  than  litmus)  the  reaction  of  the  medium  will  be  -(-1.5 
(acid  1.5  per  cent. — that  is  to  say,  each  one  hundred  cubic  centimeters  of  the  medium 
contains  a  sufficient  excess  of  acid  elements  to  require  for  their  neutralization  1.5  cubic 
centimeters  of  a  normal  sodium  hydroxide  solution).  Although  this  is  accepted  as  a 
standard  reaction,  it  will  not  infrequently  be  observed  that  variations  presenting 
greater  or  less  percentage  of  acid,  or  which  are  actually  alkaline,  will  be  more  favorable 
for  the  growth  of  individual  forms  of  bacteria ;  and  a  certain  number  of  such  modified 
media  may  be  advantageously  kept  in  stock  for  special  work.  The  reaction  is  to  be 
indicated  by  the  percentage  quantity  of  acid  or  alkali  (hydrochloric  acid  or  sodium 
hydrate)  in  normal  solution  required  for  the  neutralization  of  one  hundred  cubic  centi- 
meters of  the  medium,  the  mark  -f  indicating  acid,  the  mark  — ,  alkaline  reaction. 
Thus,  bouillon,  sufficiently  acid  to  require  for  the  neutralization  of  one  hundred  cubic 
centimeters  0.5  cubic  centimeter  of  a  normal  solution  of  sodium  hydroxide,  would  be 
indicated  as  -f  0.5  ;  a  bouillon  of  alkaline  reaction  sufficient  to  require  for  the  neutraliza- 
tion of  one  hundred  cubic  centimeters  0.75  cubic  centimeter  of  a  normal  solution  of 
muriatic  acid  would  be  represented  as  — 0.75  reaction.  For  the  determination  and  ad- 
justment of  reaction  there  will  be  required  the  following  solutions:  (a)  a  normal  solu- 
tion of  sodium  hydroxide  in  distilled  water  (40  grams  NaOH,  1000  cubic  centimeters 
water) ;  (6)  a  decinormal  solution  of  sodium  hydroxide  (4  grams  to  1000  cubic  centimeters 

70 


72  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

distilled  water) ;  (c)  a  decinormal  solution  of  hydrochloric  acid  (3.65  grams  to  1000  cubic 
centimeters  distilled  water) ;  (d)  a  normal  solution  of  hydrochloric  acid  (36.5  grams  to 
1000  cubic  centimeters  distilled  water) ;  (e)  a  0.5  per  cent,  solution  of  phenolphthalein 
in  fifty  per  cent,  alcohol.  In  the  reaction-correction  of  a  given  medium  (supposing  it 
to  be  excessively  acid),  the  volume  is  first  to  be  corrected  to  a  definite  amount.  This  is 
most  readily  done  by  correction  of  weight,  the  weight  of  a  given  volume  of  the  medium 
having  been  previously  determined  by  practice  or  calculation  for  the  temperature  of  the 
room  (20°  C.).  If  the  weight  be  less  than  it  should  be,  water  is  to  be  added  to  the  re- 
quired amount ;  if  excessive,  the  excess  is  to  be  evaporated  by  boiling.  This  attended 
to,  five  cubic  centimeters  (or  the  equivalent  weight,  preferably)  are  withdrawn,  forty- 
five  cubic  centimeters  of  distilled  water  added,  and  this  boiled  for  several  minutes 
in  a  porcelain  evaporating  dish  to  expel  any  carbon  dioxide  present.  One  cubic  centi- 
meter of  the  phenolphthalein  solution  is  now  added,  and  the  mixture  titered  while  hot 
with  the  decinormal  sodium  hydroxide  solution  from  a  burette  provided  with  a  glass 
stop-cock.  The  alkaline  solution  is  added  slowly,  drop  by  drop,  with  constant  stirring, 
until  a  distinct  pink  color  results.  The  number  of  cubic  centimeters  used  of  the  deci- 
normal sodium  hydroxide  solution  is  now  read  from  the  burette  markings,  indicating 
the  quantity  of  solution  of  this  strength  necessary  for  the  complete  neutralization  of  five 
cubic  centimeters  of  the  medium.  If  there  were  originally  1000  cubic  centimeters  of  the 
medium,  995  cubic  centimeters  would  remain;  and  for  the  complete  neutralization  of 
this  amount  the  quantity  of  decinormal  sodium  solution  can  readily  be  calculated. 
Addition  of  so  large  an  amount  of  fluid  as  this  would  entail  would,  however,  be  compli- 
cating in  its  effect ;  and  to  avoid  this,  one-tenth  the  same  quantity  of  a  normal  solution 
is  substituted.  However,  the  adopted  standard  is  not  the  absolute  neutral  point,  but 
a  reaction  of  such  acidity  as  to  require  for  the  complete  neutralization  1.5  cubic  centi- 
meters of  normal  sodium  hydroxide  .solution  for  each  one  hundred  cubic  centi- 
meters of  the  remaining  medium.  Therefore,  from  the  total  quantity  of  the  normal 
alkaline  solution  calculated  to  be  required  for  the  complete  neutralization  of  the  remain- 
ing 995  cubic  centimeters  of  the  medium  are  to  be  deducted  14.9  (9.95  by  1.5)  cubic 
centimeters;  the  remaining  number  of  cubic  centimeters  of  the  normal  alkaline 
solution  are  then  to  be  added  and  diffused  to  render  the  medium  of  the  standard  (+1.5) 
reaction.  If  the  medium  was  originally  alkaline,  on  the  addition  of  the  phenolphthalein 
it  should  have  been  titered  in  the  same  way  with  a  decinormal  solution  of  hydrochloric 
acid  until  the  discharge  of  its  pink  color.  The  number  of  cubic  centimeters  thus 
used  is  employed  as  basis  for  calculation  of  the  quantity  of  the  same  strength  acid  solu- 
tion necessary  for  the  neutralization  of  the  remaining  medium.  One-tenth  this  amount, 
plus  1.5  cubic  centimeters  additional  for  each  one  hundred  cubic  centimeters  of  the 
remaining  medium,  is  added  in  the  form  of  the  normal  solution  of  the  acid  in  order  to 
produce  the  standard  reaction. 


I.  CARBOHYDRATE    MEDIA. 

i .  Potatoes. — These  vegetables  are  most  frequently  employed  as  a  culture  medium 
for  the  chromogenic  bacteria  and  for  a  few  other  organisms,  as  those  of  typhoid  fever  and 
of  glanders,  which  produce  rather  characteristic  growths  upon  them.  Their  preparation 
is  simple,  consisting  merely  of  proper  cleansing,  cooking,  and  sterilization,  inoculations 
being  made  upon  the  cut  surface  of  the  vegetable.  It  is  not  customary  to  modify  the 
natural  (acid)  reaction  of  the  potato  in  such  use.  They  may  be  employed  either  in  dish 
or  tube  cultures,  preferably  the  latter. 


74 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


(a)   Dish  Cultures. — 

Exercise  23. — Select  potatoes  of  as  regular  shape  as  possible,  without 
blemish  and  with  as  few  eyes  as  possible,  each  student  preparing  two 
potatoes.  Wash  them  in  running  water,  using  a  stiff  hand  brush  for 
effectiveness.  The  eyes  and  any  suspicious  spots  are  next  cut  out  freely, 
after  which  the  potatoes  are  immersed  in  a  dish  containing  i :  1000  solu- 
tion of  bichloride  of  mercury  for  an  hour.  They  are  then  put  in  the  steam 
sterilizer  in  a  tin  bucket  with  perforated  bottom  ("potato  bucket")  and 
cooked  for  three-quarters  of  an  hour  or  an  hour  at  100°  C.  They  are 
not  to  be  removed  from  the  sterilizer,  but  are  again  steamed  at  the  same 
temperature  for  fifteen  minutes  upon  the  second  and  third  days.  A 
potato  dish  should  in  the  mean  while  be  prepared  for  their  recep- 
tion at  the  close  of  the  process 
of  sterilization  upon  the  third  day 
(i>.  instructions,  Ex.  22).  The 
hands  of  the  operator  are  now  to 
be  thoroughly  washed  with  hot 
water,  soap,  and  brush,  and  soaked 
for  five  minutes  in  a  i :  1000  solu- 
tion of  mercuric  chloride  and  a 
potato  knife,  a  flat-bladed  knife 
such  as  is  commonly  sold  for  use 
in  the  household  for  paring  pota- 
toes (Fig.  22),  is  sterilized  by  flam- 
ing (i>.  instructions,  Ex.  6)  if  it  has 
not  previously  been  prepared  by 
baking  or  boiling.  The  opera- 
tor, holding  the  potato  with  the  thumb  and  fingers  of  the  left  hand 
grasping  its  shortest  diameter,  divides  it  into  halves,  drawing  as  little 
of  the  blade  as  possible  through  it  and  retaining  the  blade  in  position 
between  the  cut  surfaces  without  separating  the  two  halves.  An  assistant 
raising  the  cover  of  the  culture  dish,  the  potato  is  deposited  therein  in 
position  so  that,  as  the  two  halves  are  separated  by  proper  movement 
of  the  knife,  the  cut  surfaces  will  fall  apart  and  remain  uppermost.  The 
dish  is  now  temporarily  closed,  and  the  second  potato  is  in  like  manner 
to  the  first  divided  and  placed  in  the  dish  with  its  cut  surfaces  exposed. 
In  these  operations  no  more  exposure  of  the  interior  of  the  dish  is  allowed 
for  entrance  of  organisms  from  the  air  than  can  be  avoided ;  and  obviously 
the  procedure  should  not  be  performed  where  any  atmospheric  draughts 
prevail.  In  order  that  the  student  may  obtain  some  idea  of  the  objec- 
tionable features  of  such  cultures  and  the  value  of  the  method,  this  first 


FIG.  22. — SECTION  OF  POTATO  INTENDED  FOR 
DISH  CULTURE. 


76 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


dish  may  profitably  be  left  closed  and  uninoculated  for  the  purpose  of 
observation.  It  is  very  common  that  in  spite  of  all  care  exercised  the 
potatoes  soon  show  the  growth  of  numerous  colonies,  infection  having 
taken  place  from  the  air  which  was  inclosed  in  the  dish  (unsterilized  air)  at 
some  time  when  the  cover  was  removed,  or  entered  the  closed  dish  through 
unobserved  imperfections  in  the  line  of  application  of  the  cover  to  the 
edge  of  the  dish  (should  slight  currents  be  in  some  way  induced  through 
such  small  apertures).  This  last  possible  method  of  convection  of  infec- 
tion may  be  prevented  by  applying  a  thick  layer  of  vaseline  to  the  margin 
of  the  dish  before  applying  the  cover. 

(6)  Potato  Tubes. — 

Exercise  24. — One  dozen  potato  tubes  have  been  previously  prepared 
and  once  heated  in  the  oven  (Ex.  22).  The  same  care 
as  to  the  selection  of  suitable  potatoes  is  to  be  observed 
as  in  the  preceding  exercise.  They  are  then  carefully 
washed  and  rinsed  in  running  water.  The  ends  of  each 
potato  are  cut  squarely  off,  and  with  a  cork-borer  of 
a  diameter  slightly  less  than  that  of  the  tubes  (or  with 
a  knife  if  the  cork-borer  be  not  at  hand),  cylinders  of 
the  potato  substance  are  cut  from  each  and  placed 
in  water.  After  half  a  dozen  such  cylinders  have  been 
obtained,  each  is  to  be  cut  in  an  oblique  fashion  so 
that  each  resulting  piece  presents  a  round,  flat  end 
and  a  large  oval  beveled  surface  for  exposure  in  the 
tube  (Fig.  23).  These  pieces  are  now  left  in  running 
water  for  several  hours  (or  over  night  if  convenient), 
the  washing  preventing  their  discoloration  in  the  sub- 
sequent sterilization.  Thereafter  each  bit  is  intro- 
duced into  a  tube,  beveled  surface  uppermost,  the  flat 
end  resting  on  the  bit  of  glass  rod  (or  wad  of  cotton  or 
other  rest)  placed  in  the  bottom  of  the  tube,  and  the 
cotton  stopper  is  readjusted.  When  all  the  tubes  are  thus  filled,  they 
are  placed  in  the  steam-bath  (at  100°  C.  for  thirty  minutes,  repeated 
for  fifteen  minutes  on  the  second  and  third  days)  or  in  the  autoclave 
(120°  C.  for  thirty  to  forty  minutes)  for  sterilization. 

(c)  Occasionally,  after  the  potatoes  are  washed  and  pared  they  are  cut  into  broad, 
thin  slices,  which  are  placed  in  Petri  dishes  and  sterilized  there  just  as  if  in  tubes.  In 
this  way  extensive  surface  exposure  may  be  obtained,  of  material  advantage  in  the 
separation  of  mixed  cultures. 

Further,  potatoes,  after  being  cleansed  and  boiled,  may  be  mashed  and  the  result - 


FIG.  23. — CULTURE 
TUBE  CONTAIN- 
ING CYLINDER  OF 
POTATO  RESTING 
ON  SMALL  GLASS 
ROD. 


78  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

ant  paste  may  be  spread  in  dishes  or  introduced  into  tubes  if  desired.  In  this  method 
of  preparation  one  may  take  advantage  of  the  opportunity  afforded  for  adjusting  the 
reaction  of  the  medium,  by  boiling  the  paste  in  a  sodium  hydrate  solution  to  correct  the 
reaction  uniformly  through  the  mass. 

Glycerine  potato  tubes  differ  from  the  above  ordinary  tubes  merely  in  that  a  five  per 
cent,  solution  of  glycerine  is  introduced  into  the  tube  before  sterilization,  up  to  the  level 
of  the  lower  surface  of  the  potato ;  from  which  a  small  amount  of  the  glycerine  diffuses 
gradually  through  the  potato  and  over  its  surface.  Tubertle  bacilli  may  be  grown  upon 
such  a  preparation. 

(d)  Eisner's  Medium. — This  is  a  modification  of  Holz's  potato  gelatine  (used  for  the 
same  purpose  as  Eisner's  medium),  designed  as  an  advantageous  medium  for  the  isola- 
tion of  the  Bacillus  typhosus  and  Bacillus  coli  communis  from  the  variety  of  organisms 
with  which  they  are  apt  to  be  associated  in  the  dejecta,  and  from  each  other.  Upon 
it  the  common  saprophytic  germs  are  almost  completely  inhibited,  and  the  typhoid 
fever  organism  develops  at  a  later  period  than  Bacillus  coli  and  with  such  differences  of 
appearance  in  the  colonies  as  to  make  possible  the  distinction  of  one  from  the  other, 
thus  facilitating  their  separation.  The  medium  may  be  made  as  follows:  Five  hundred 
grams  of  pared  potato  are  grated  finely  and  the  pulp  placed  in  the  refrigerator,  in  a  porce- 
lain dish,  over  night.  The  following  morning  the  juice  is  expressed  from  the  pulp  and 
filtered  several  times  through  a  layer  of  absorbent  cotton  or  through  animal  charcoal 
(preferably  the  latter).  The  filtrate  should  now  be  titered  with  decinormal  sodium 
hydroxide  solution  to  determine  its  reaction  and  the  amount  of  water  which  will  be 
required  to  be  added  to  reduce  its  acidity  to  the  standard.  In  this  quantity  of  water  is 
now  boiled  the  amount  of  gelatine  required  to  make  a  ten  per  cent,  proportion  of  the 
gelatine  when  the  potato  juice  shall  have  been  added,  the  reaction  of  the  gelatine  being 
corrected  to  neutral  point  after  it  has  been  dissolved  (and  before  adding  the  potato 
juice)  by  means  of  normal  sodium  hydroxide  solution.  This  done,  and  the  bulk  of  the 
fluid  corrected  for  evaporation  to  that  necessary  to  properly  dilute  the  potato  juice,  the 
latter  is  slowly  added  to  the  gelatine  and  mixed,  and  the  whole  boiled  for  five  to  ten 
minutes,  filtered,  and  distributed  to  tubes  or  flasks,  and  sterilized  in  the  usual  inter- 
rupted manner  in  steam.  Thus  far  the  medium  constitutes  Holz's  potato  gelatine. 
Eisner's  medium  is  made  from  this  by  adding  freshly  when  required  for  use  one  cubic 
centimeter  of  a  sterilized  solution  (ten  per  cent,  strength)  of  potassium  iodide  to  each 
ten  cubic  centimeters  of  the  medium.  It  will  be  found  advantageous  for  incubation  at 
body  heat  that  one-half  the  required  amount  of  gelatine  be  substituted  by  a  correspond- 
ing amount  of  agar  (one  per  cent,  of  agar). 

2.  A  number  of  other  carbohydrate  media  are  employed  for  culture  purposes,  as  car- 
rots, turnips,  apples,  bread-paste,  etc.  They  are  used  for  the  most  part  for  the  cultiva- 
tion of  the  moulds  and  of  chromogenic  forms  of  bacteria,  but  are  scarcely  of  sufficient, 
importance  to  be  further  detailed  here.  The  various  sugars  as  culture  media  will  be 
noted  in  connection  with  the  media  in  which  they  are  usually  used. 


II.   PROTEID    MEDIA. 

By  experience  it  has  been  found  that  nutrient  media  containing  proteid  substance 
are  upon  the  whole  the  most  favorable  for  the  culture  of  the  greatest  number  of  known 
bacteria,  particularly  the  pathogenic  forms ;  and  it  is  probable  that  with  further  study 
the  selection  of  such  material  will  be  far  more  exact  than  at  present  apprehended.  The 
usual  source  at  present  is  adult  beef ;  yet  it  is  probable,  from  the  comparatively  limited 


80  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

experience  of  bacteriologists  in  this  direction,  that  much  can  be  done  in  selective  cultiva- 
tion by  substituting  the  proteids  of  other  animals,  even  from  man.  The  development  of 
this  field  presents  possibilities  for  investigation.  The  albumins  at  present  utilized  are 
for  the  most  part  of  the  nature  of  albumoses,  peptones,  and  serum  albumin ;  and  their 
preparation  is  largely  empiric,  and  the  precise  composition  of  the  products  a  matter  of 
uncertainty.  The  amount  of  albuminous  substance  in  ordinary  bouillon  and  its  deriva- 
tives is  in  reality  much  less  than  was  originally  contemplated  in  its  manufacture ;  and  it 
is  probable  that  the  many  complex  organic  and  inorganic  salines  and  derivatives  pre- 
served in  its  manufacture  constitute  the  most  important  feature  of  the  substance.  One 
sees  but  little  difference  practically,  for  example,  in  the  cultural  qualities  of  the  media 
made  from  the  commercial  beef  extracts  and  those  made  from  the  fresh  beef ;  yet  it  is 
well  known  that  these  extracts  contain  comparatively  little  of  the  meat  albumins  and 
are  largely  composed  of  the  various  salines  and  extractives.  In  blood-serum,  of  course, 
the  albumin  of  the  serum  is  preserved ;  and  this  substance  more  nearly  than  the  bouillon 
preparations  represents  a  natural  proteid  medium.  In  bouillon  preparations  the  loss 
of  the  albumins  is  sought  to  be  corrected  by  the  addition  of  commercial  peptones  (really 
albumose  for  the  most  part) ;  and  solutions  of  the  latter  will  be  found  to  possess  every 
practical  value  of  the  more  complexly  arranged  bouillon. 

i.  Bouillon. — Bouillon  is  the  nutrient  medium  commonly  selected  when  large, 
massive  cultures  of  bacteria  are  required  to  be  grown,  or  when  it  is  desired  to  have 
the  bacteria  in  liquid  surroundings,  to  facilitate  observations  as  to  their  power  of 
movement,  their  intimate  relations,  or  their  chemical  activities;  or  when  they  are 
intended  for  animal  inoculation.  It  is  prepared  either  from  the  fresh  meat  or  from 
the  commercial  beef  extracts.  It  is  very  much  more  convenient  to  make  the  prepara- 
tion from  the  latter,  and  so  far  as  the  value  of  the  product  as  a  nutrient  medium  is 
concerned,  it  is  apparently  in  no  way  less  advantageous  than  that  made  from  the 
fresh  meat.  It  possesses  one  important  advantage,  moreover,  in  that  it  is  less  likely 
to  contain  the  meat  sugars  uniformly  met  in  the  meat  bouillon,  rendering  it  preferable 
in  exercises  intended  to  demonstrate  reaction  changes  and  gas  formation  by  bacteria ; 
and  in  its  manufacture  it  is  more  easy  to  maintain  a  definite  and  uniform  composi- 
tion. 

(a)  When  making  bouillon  from  the  fresh  beef,  the  meat  is  obtained  from  the 
butcher  as  fresh  as  possible ;  it  should  be  free  from  fat  and  should  be  finely  chopped 
or  ground.  The  meat  pulp  thus  procured  is  soaked  over  night  in  water  (preferably 
boiled  water — in  proportion  of  500  grams  of  the  pulp  to  1000  cubic  centimeters  of 
water),  being  kept  in  the  refrigerator  in  order  to  prevent  the  development  of  the  bac- 
teria present  in  the  meat.  The  following  morning  the  red  meat  infusion  is  strained 
off  through  a  piece  of  cheese-cloth,  the  pulp  being  compressed  if  necessary  to  obtain 
a  full  liter  of  the  liquid.  If  less  than  a  liter  be  obtainable,  it  is  customary  to  add 
water  to  make  up  the  amount  to  this  quantity.  The  infusion  is  next  heated  moder- 
ately (between  50°  and  60°  C.)  in  a  suitable  vessel  (as  a  granite-ware  stew-pan),  and 
ten  grams  of  dried  peptone  and  five  grams  of  sodium  chloride  are  added,  and  with  con- 
stant stirring  with  a  clean  glass  rod,  thoroughly  mixed  and  dissolved  in  the  warm  infu- 
sion. When  the  peptone  and  salt  are  dissolved,  the  preparation  is  boiled  sharply,  with 
constant  stirring,  until  all  the  albumins  coagulable  by  heat  are  separated  and  the  liquid 
is  of  a  clear,  amber  color.  It  is  then  filtered  through  paper;  and  should  the  filtrate 
be  at  all  turbid,  it  should  be  reboiled  and  again  filtered.  The  filtrate  is  now  cooled 
to  room  temperature  and  the  amount  corrected  to  one  liter,  after  which  it  is  titered 
with  decinormal  sodium  hydroxide  solution  and  the  reaction  adjusted.  It  is  again 


82  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

to  be  boiled  and  filtered,  after  which  it  may  be  distributed  to  tubes  or  flasks  and  ster- 
ilized by  the  fractional  method  in  steam  (100°  C.),  or  in  the  autoclave  (120°  C.). 

(6)  When  the  bouillon  is  to  be  made  from  beef  extract,  1000  cubic  centimeters 
of  water  are  placed  in  a  clean  stew-pan,  and  five  grams  (various  proportions  have 
been  recommended,  from  two  to  five  grams)  of  Liebig's,  or  other  standard  brand 
of  extract,  added,  together  with  ten  grams  of  dried  peptone  and  five  grams  of  sodium 
chloride.  Gentle  heat  is  applied  to  facilitate  solution,  the  preparation  being  con- 
stantly stirred  with  a  clean  glass  rod.  When  solution  has  been  effected,  the  liquid 
is  well  boiled  for  from  five  to  ten  minutes,  with  constant  stirring.  It  is  then  filtered 
through  paper,  cooled,  and  the  volume  corrected  to  one  liter,  after  wrhich  the  reaction 
is  determined  and  adjusted  in  the  usual  manner.  It  is  thereafter  again  boiled  and 
filtered,  and  distributed,  when  it  is  sterilized  in  the  usual  manner. 

Exercise  25. — Each  student  prepare  one  liter  of  bouillon  from  beef 
extract  as  above  directed,  dividing  the  product  as  follows :  500  cubic  centi- 
meters for  use  in  the  manufacture  of  gelatine  and  agar;  300  cubic  centi- 
meters for  manufacture  of  glucose-,  lactose-,  and  saccharose-bouillon; 
and  200  cubic  centimeters  for  use  as  plain  bouillon,  distributing  of  the 
last,  sixty  cubic  centimeters  to  a  dozen  tubes,  and  placing  the  remainder 
in  a  stock-flask  for  future  distribution  as  needed.  Measure  out  1000  cubic 
centimeters  of  distilled  water  into  a  clean  granite-ware  stew-pan.  Add 
five  grams  of  Liebig's  extract,  which  has  been  weighed  out  on  a  balanced 
slip  of  paper  (paper  and  contents  thrown  into  the  water),  ten  grams  of 
dried  peptone  (Witte's),  and  five  grams  of  table  salt.  Warm  and  stir 
until  the  above  ingredients  are  dissolved.  Boil  for  five  to  ten  minutes 
to  separate  any  coagulable  albumins.  Cool  to  room  temperature  and 
correct  volume  to  original  amount  by  addition  of  boiled  distilled  water. 
Determine  reaction  and  adjust  to  standard.  Heat  by  strong  boiling  or 
in  autoclave  for  ten  or  fifteen  minutes,  and  allow  to  cool  for  separation 
of  excess  of  phosphates.  Filter  and  distribute,  and  sterilize  by  fractional 
method  in  steam. 

(c)  Glucose-,  Lactose-,  and  Saccharose-bouillon. — These  preparations  are  used  mainly 
for  the  determination  of  fermentative  qualities  of  various  organisms,  the  destruction 
of  the  carbohydrates  setting  free  carbon  dioxide.  For  their  manufacture  a  bouillon 
known  to  be  free  from  meat  sugar  should  be  prepared,  to  which  is  added  one  per  cent, 
of  glucose  for  glucose-bouillon;  one  per  cent,  of  lactose  for  lactose-bouillon;  or  the 
same  proportion  of  saccharose  for  saccharose-bouillon.  The  presence  of  meat  sugar 
in  a  bouillon  may  be  determined  by  testing  with  Fehling's  solution  or  by  inoculating 
a  fermentation  tube  filled  with  the  sample  to  be  tested,  with  a  gas-forming  organism, 
like  the  Bacillus  coli,  incubating  it  at  body  temperature  for  twenty-four  hours,  when 
gas  formation  will  be  found  to  have  taken  place  if  sugar  be  present  in  the  bouillon. 
Usually,  bouillon  made  from  beef  extract  is  free  from  this  substance  and  is  therefore 
of  advantage  in  the  preparation  of  these  media.  If  one  will  inoculate  a  flask  of  ordinary 
bouillon  with  the  Bacillus  coli  communis  and  allow  it  to  grow  for  several  days  at  in- 
cubator temperature,  the  sugar  will  be  destroyed  thereby ;  and  by  subsequent  steriliza- 


84  LABORATORY  EXERCISES  IN  BACTERIOLOGY, 

tion  and  filtration  the  bouillon  will  be  rendered  suitable  for  the  preparation  of  these 
special  media. 

Exercise  26. — Of  the  bouillon  made  by  the  student  in  the  preceding 
exercise,  after  testing  with  Fehling's  solution  to  determine  that  no  sugar 
is  present,  withdraw  three  portions  of  one  hundred  cubic  centimeters 
each,  the  amount  retained  for  the  preparation  of  the  sugar  bouillons. 
(If  these  amounts  be  withdrawn  from  a  general  stock,  the  remaining  por- 
tion is  to  be  resterilized.)  Each  of  these  portions  is  to  be  placed  in  a 
clean  beaker  or  flask,  and  one  gram  of  glucose  added  to  the  first  and  a 
like  amount  respectively  of  lactose  and  saccharose  to  the  second  and  third. 
Aided  by  gentle  warming  and  stirring  with  a  clean  glass  rod,  these  sub- 
stances are  dissolved,  and  the  product  placed  in  fermentation  tubes  or 
retained  in  the  flasks,  and  sterilized. 

DISTRIBUTION  OF  LIQUID  MEDIA  TO  TUBES,  ETC. 

The  distribution  of  fresh  blood-serum,  bouillon,  peptone  solution,  and  other 
nutrients  liquid  at  ordinary  temperatures,  and  of  gelatine  and  agar  preparations 
after  liquefaction  by  heat,  is  performed  in  the  same  way  whether  the  destination  of 
the  material  be  to  tubes,  flasks,  or  dishes.  In  case  of  blood-serum  intended  for  the 
preparation  of  a  clear  jelly,  the  utmost  care  must  be  taken  to  avoid  contamination 
by  microorganisms,  since  in  the  further  steps  of  its  preparation  it  will  not  be  possible 
to  depend  certainly  upon  the  low  degree  of  heat  alone  permissible  for  sterilization. 
In  case  of  gelatine,  too,  inasmuch  as  this  substance,  if  overheated  or  heated  too  fre- 
quently or  too  long,  is  liable  to  alterations  and  loss  of  its  power  of  setting,  caution 
should  be  exercised  to  prevent  infection  of  the  finished  preparation.  With  the  other 
forms  of  nutrient  media,  including  blood-serum  intended  for  the  opaque  white  solid 
serum,  there  need  not  be  particular  care  in  the  process.  When  small  quantities  are 
to  be  distributed,  probably  the  most  convenient  method  will  be  found  in  the  withdrawal 
of  the  liquid  or  liquefied  medium  from  the  stock-flask  by  means  of  a  pipette  of  fifty 
to  one  hundred  cubic  centimeters  capacity  (if  convenient  the  pipette  should  be  sterile, 
but  this  is  not  essential).  It  is  convenient  here  that  an  assistant  should  hold  the 
tubes,  withdraw  the  cotton  stoppers  for  introduction  of  the  medium,  and  thereafter 
replace  the  same  in  the  tubes.  However,  a  stand  can  easily  be  made  by  boring  holes 
of  suitable  diameter  and  depth  into  a  block  of  wood,  to  serve  as  a  stand  for  the  tubes 
in  the  process,  which  can  then  be  readily  conducted  by  one  person.  In  introducing 
the  medium  into  the  tubes  it  should  not  be  permitted  to  come  in  contact  with  the 
upper  part  of  the  inner  surface,  lest  after  the  application  of  the  cotton  stopper  the 
latter  may  be  contaminated  and  perhaps  glued  to  the  glass. 

A  convenient  method  is  to  distribute  from  a  separation  funnel  (or  an  ordinary 
funnel  covered  by  a  plate  to  prevent  contamination,  as  shown  in  figure  24),  a  rubber 
tube  fitted  with  a  pinch-cock  connecting  a  glass  pipette  to  the  lower  end  of  the  funnel. 

If  care  is  taken  there  is  no  objection  to  pouring  the  medium  from  the  flask  into 
the  tubes,  first  having  flamed  the  lip  of  the  flask  to  destroy  bacteria  which  are  apt 
to  be  lodged  upon  it. 

Where  great  caution  is  to  be  observed,  as  in  the  distribution  of  fresh  blood-serum, 
siphonage  is  to  be  recommended,  the  siphon  arranged  as  shown  in  the  accompanying 


86 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


diagram  (Fig.  25).  The  principle  of  the  ordinary  chemical  wash-bottle,  too,  may  be 
easily  utilized  for  distribution  from  stock-flasks,  the  tube  carrying  the  compressed 
air  into  the  flask  being  provided  with  a  cotton  plug  to  prevent  the  entrance  of  con- 
taminating organisms,  and  force  being  applied  from  any  convenient  source,  as  a  reversed 
siphon,  compressed  air  tank,  carbonic  oxide  tank,  or  from  the  mouth  (Fig.  26).  Of 
course,  in  all  cases  in  which  it  is  essential  to  prevent  contamination  the  tube  of  the  siphon 
or  other  appliance  introduced  into  the  medium  should  have  been  properly  sterilized. 

The  distribution  flasks  devised  by  various  workers,  several  of  which  are  shown 
in  figure  17,  are  convenient,  but  by  no  means  essential. 

In  tubing  it  is  customary  to  put  about  five  cubic  centimeters  of  medium  into 


FIG.  24.— DISTRIBUTION  OF  LIQUID  MEDIUM 
FROM  COVERED  FUNNEL  TO  CULTURE 
TUBE. 


FIG.  25. 


each  tube,  approximation  of  this  amount  being  sufficient  for  ordinary  work.  It  is 
well,  however,  to  provide  a  number  of  tubes  containing  exact  amounts,  for  special 
purposes.  For  dilution  experiments  four  or  nine  cubic  centimeters  will  be  found 
convenient  quantities.  Tubes  of  the  same  size  having  been  selected,  the  desired 
number  of  centimeters  of  water  are  placed  in  one  tube,  and  from  the  level  of  this 
the  other  tubes  are  marked  and  the  medium  subsequently  filled  in  to  the  mark  upon 
each. 

2.  Peptone  Gelatine  ("Gelatine"). — This    medium    consists  of    the    ordinary 
bouillon  to  which  has  been  added  from  ten  to  fifteen  per  cent,  of  the  best  sheet  gelatine 


88 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


in  order  to  give  it  a  solid  consistence.  The  especial  value  of  a  solid  over  a  liquid  medium 
rests  in  the  fact  that  it  does  not  conduce  to  the  free  diffusion  of  the  bacteria  inoculated 
upon  it,  but  permits  their  development  into  definite  and  more  or  less  isolated  colonies, 
presenting  gross  characteristics  apt  to  be  lost  by  diffusion  through  a  liquid,  thus  facili- 
tating the  recognition  of  the  organisms  and  their  separation  into  pure  cultures.  There 
is  comparatively  little  nutrient  value  in  the  gelatine,  but  it  affords  an  additional  value 
in  being  liquefied  by  the  proteolytic  power  of  a  large  class  of  bacteria,  thus  giving 
rise  to  a  broad  classification  of  these  microorganisms  into  the  two  great  classes  of 
liquefying  and  non-liquefying  bacteria.  The  solid  consistence  of  gelatine  is  lost  at 
about  30°  C.,  and  its  usefulness  is  therefore  impaired  for  bacteria  requiring  incubation 
at  a  temperature  above  this.  This  disadvantage  may  be  obviated  in  some  measure 
by  combining  with  it  a  small  amount  of  agar.  In  the  preparation  of  gelatine  it  should 
be  kept  in  mind  that  superheating  or  too  prolonged  or  too  frequent  heating,  even 
at  the  ordinary  temperature  of  the  steam-bath  (100°  C.),  may  convert  the  gelatine 


FIG.  26. — DISTRIBUTION  TO  CULTURE  TUBE  OF   LIQUID   MEDIUM   FROM   FLASK   A,  BY 
PRESSURE  FROM  SIPHON  FLASK  B. 


into  paragelatine  and  destroy  its  power  of  congelation ;  and  caution  in  this  connection 
is  essential  for  the  best  results.  In  its  manufacture,  if  it  be  necessary  to  freshly  prepare 
the  bouillon,  the  process  may  be  shortened  by  neutralizing  it  immediately  after  solu- 
tion of  the  peptone  and  salt  (litmus  paper  may  in  this  preliminary  neutralization  be 
used  as  a  sufficiently  exact  indicator),  and  then  adding  the  gelatine.  When  prepared, 
tubed,  and  sterilized,  those  tubes  intended  for  surface  inoculation  should  be  placed 
in  a  slanting  position,  so  that  the  medium  may  set  with  a  larger  surface  for  exposure. 
The  congelation  is  best  secured  by  rapid  cooling  of  the  medium  on  a  block  of  ice,  or 
in  the  refrigerator,  or  in  a  bath  of  ice-water. 

Exercise  27. — (Prepare  bouillon  as  just  suggested  if  not  on  hand.) 
Two  hundred  and  fifty  cubic  centimeters  of  the  bouillon  prepared  in 
exercise  25  are  measured  into  a  clean  stew-pan.  Add  twenty-five  grams 
of  the  best  sheet  gelatine,  broken  or  cut  into  small  pieces,  and  heat  gently 


90  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

to  dissolve.  When  the  gelatine  has  been  dissolved,  cool  the  preparation 
to  60°  C.  or  less,  and  add  thereto  one-half  the  white  of  an  egg  dissolved 
in  twenty-five  cubic  centimeters  of  boiled  water  (ten  per  cent,  solution 
of  commercial  egg-albumen  may  be  substituted),  mixing  it  well  with 
the  liquid  by  stirring.  Boil  for  ten  or  fifteen  minutes  to  coagulate  the 
egg-albumen  and  to  reduce  the  volume  by  evaporation  to  the  original 
amount  or  less.  Filter  hot  through  a  moistened  filter  paper  which  has 
been  folded  after  the  manner  customary  in  the  chemical  laboratory,  to 
separate  the  coagulated  egg-albumen  (albumen  added  and  coagulated 
in  order  that  solid  particles  which  might  interfere  with  the  transparency 
of  the  product  may  be  caught  in  the  meshes  of  the  coagulum  and  thus 
be  removed) ;  correct  volume  (correct  to  original  weight,  i.  e,,  weight  of 
bouillon  plus  weight  of  gelatine).  While  yet  hot  determine  reaction  and 
adjust  to  standard.  If  medium  becomes  clouded,  again  boil  and  filter. 
Distribute  half  to  tubes,  and  sterilize  by  fractional  steaming;  retain  re- 
mainder in  small  stock-flask  and  sterilize  in  the  same  manner. 

3.  Peptone  Agar-agar  ("Agar"). — This  medium  is  made  by  the  addition  of 
two  per  cent,  of  agar-agar  (a  vegetable  gelatine  derived  from  an  alga  found  on  the 
Eastern  Asiatic  coast)  to  bouillon.  The  purpose  of  the  addition  of  this  substance 
is  the  same  as  that  of  the  addition  of  gelatine — i.  e.,  the  solidification  of  the  mass; 
but  as  the  medium  after  preparation  is  not  liquefied  by  temperatures  below  85°  to 
90°  C.  (again  setting  at  about  40°  C.),  it  is  better  suited  than  gelatine  for  culture  of 
organisms  requiring  incubation.  In  the  manufacture  it  is  necessary  that  the  agar 
should  have  been  thoroughly  dissolved  into  a  limpid  liquid  for  ready  filtration ; 
this  is  accomplished  by  prolonged  boiling  (one  or  two  hours),  its  property  of  con- 
gelation not  being  interfered  with  by  such  exposure  to  heat.  The  presence  of  excess 
of  acid  in  the  material  to  which  the  agar  is  added  is  more  or  less  harmful  to  its  solidi- 
fying power,  for  which  reason  the  bouillon  should  have  been  neutralized  (litmus  re- 
action sufficient)  before  the  agar  is  added.  The  product  is  not  quite  so  clear  as  gelatine 
at  best.  This  medium  is  not  affected  by  the  proteolytic  ferments. 

Exercise  28. — Five  grams  of  finely  chopped  or  ground  agar  are  boiled 
for  one  or  two  hours  in  one  hundred  cubic  centimeters  of  water  in  a  beaker 
or  other  vessel,  water  being  added  from  time  to  time  to  prevent  drying. 
To  this  is  added  at  the  close  of  boiling  250  cubic  centimeters  of  the  bouillon 
prepared  in  exercise  25  (or  same  amount  of  bouillon  freshly  prepared 
up  to  the  stage  of  sterilization,  if  stock  bouillon  be  not  on  hand) ;  and 
the  mixture  boiled  until  evaporation  has  reduced  it  approximately  to 
the  desired  weight  (weight  of  bouillon  plus  five  grams  of  agar).  It  is 
now  cooled  to  60°  C.,  or  less,  and  one-half  the  white  of  an  egg  dissolved 
in  twenty-five  cubic  centimeters  of  boiled  water  added  and  stirred  into 
the  preparation.  Reboil  for  ten  or  fifteen  minutes  to  coagulate  the  egg- 
albumen  and  filter  through  a  folded  and  moistened  filter  paper.  If  it  is 


92 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


feared  that  filtration  will  be  slow,  the  filtration  funnel  and  receiving 
flask,  a  cover  being  adjusted  to  the  funnel  to  prevent  the  water  of  con- 
densation from  dripping  into  its  contents,  may  be  placed  in  the  auto- 
clave, where  the  temperature  is  raised  to  110°  to  115°  C.  Or  the  hot- 
water  filtration  bucket  suggested  by  Matlock  may  be  employed  to  keep 
the  preparation  well  liquefied  during  the  period  of  filtration  (Fig.  27). 
After  filtration  the  weight  should  be  exactly  corrected  (weight  of  bouillon 
plus  five  grams  of  agar)  by  evaporation  of  excess  by  further  boiling  or 
by  addition  of  boiled  water  if  deficient;  after  which  the  reaction  is  to 
be  determined  and  adjusted  to  standard.  Should  the  medium  now 
become  turbid,  it  is  to  be  reheated  and  again  filtered,  after  which  half 

of  it  may  be  transferred  to  tubes  and  the  re- 
mainder retained  in  the  stock-flask,  and  steril- 
ized either  in  the  autoclave  or  in  the  ordinary 
steam-bath  (by  fractional  steaming  in  the  lat- 
ter case).  Those  tubes  intended  for  surface 
inoculations  are  to  be  allowed  to  solidify  in 
a  slanting  position,  to  increase  surface  expo- 
sure, the  remainder  allowed  to  cool  in  erect 
position,  for  use  in  stab  cultures  and  for 
plate  cultures. 

Modifications  of  Agar. — 

(a)  Gelatine-agar .- — For  plate  cultures  agar  is 
not  as  well  suited  as  gelatine,  as  it  does  not  spread 
as  evenly  as  the  latter  unless  heated  to  a  tempera- 
ture fatal  to  bacteria  which  it  is  desired  to  have 
diffused  through  the  melted  mass;  and  on  the  other 
hand  gelatine  cannot  be  used  for  either  tube  or  plate 
cultures  if  it  be  desired  to  subject  the  cultures  to  the 

body  temperature  in  the  incubator  (37.5°  C.),  because  it  becomes  liquid  at  30°  C. 
In  order  to  adjust  these  difficulties  and  to  provide  a  medium  capable  of  indicating 
the  liquefying  power  of  bacteria  in  cultures  grown  at  incubator  temperature,  a 
mixture  of  gelatine  and  agar  has  been  proposed,  which  serves  the  desired  end 
fairly.  It  may  be  made  by  adding  the  liquefied  agar  to  liquefied  gelatine  in  equal 
proportion ;  or  in  the  manufacture  there  may  be  added  to  bouillon  but  one  per 
cent,  of  agar,  after  this  has  been  melted  adding  five  or  six  per  cent,  of  gelatine.  In 
other  respects  the  preparation  follows  the  steps  indicated  in  the  preparation  of  either 
agar  or  gelatine.  The  medium  retains  its  solid  consistence  at  incubator  temperature ; 
and  while  liquefaction  does  not  take  place  with  the  same  readiness  and  to  the  same 
degree  as  in  case  of  the  pure  gelatine,  it  occurs  sufficiently  to  be  indicative. 

(6)  Glycerine  Agar. — This  medium  is  especially  valuable  as  a  nutrient  in  the  cul- 
tivation of  tubercle  bacilli  and  a  few  other  organisms.  It  is  prepared  by  adding  five 
per  cent,  of  glycerine  to  ordinary  agar  after  it  has  been  completed,  all  but  the  steriliza- 
tion. 


FIG.   27.— HOT- WATER   FILTRA- 
TION BUCKET. 


94  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

(c)  Lactose-litmus  Agar. — This  modification  is  used  in  the  study  of  acid  production 
by  bacteria  and  as  a  means  of  differentiation  between  acid-producing  bacteria  and 
organisms  not  possessed  of  the  power  of  thus  converting  the  sugar.  It  is  prepared  by 
adding  to  agar  of  slightly  alkaline  reaction  ( — 0.5)  two  per  cent,  of  lactose,  and  after 
sterilization  tinting  the  liquid  medium  before  it  congeals  to  a  pale  blue  color  with  a 
sterile  litmus  solution.  Acid-producing  bacteria  growing  upon  this  medium  cause 
a  pinkish  discoloration  of  the  mass  in  and  about  the  colonies,  contrasting  with  the 
blue  color  of  the  rest  of  the  medium  and  the  colonies  of  bacteria  not  possessing  the 
acid-producing  power. 

4.  Peptone  Solution. — This  is  often  and  with  advantage  used  in  place  of  bouillon. 
It  is  made  by  dissolving  ten  grams  of  dried  peptone  and  five  grams  of  table  salt  in  one 
liter  of  distilled  water;  after  which  it  is  to  be  filtered,  distributed,  and  sterilized.     It 
is  not  essential  to  adjust  the  reaction,  as  it  is  almost  constantly  neutral  or  nearly  so. 
The  medium  is  especially  favorable  for  use  in  the  study  of  indol  and  phenol  production. 

Rosolic  Acid-peptone  Solution. — This  substance  serves  as  a  means  of  determining 
reaction  changes  produced  by  bacteria.  It  consists  of  the  above  peptone  solution,  to 
which  has  been  added  two  per  cent,  of  a  0.5  per  cent,  solution  of  rosolic  acid  in  eighty 
per  cent,  alcohol.  After  diffusion  of  the  dye  through  the  peptone  solution,  the  medium 
is  distributed  to  tubes  and  sterilized  by  fractional  steaming.  It  is  of  a  pale  pink 
color,  which  is  intensified  by  alkaline  changes  and  discharged  by  acid  alterations 
produced  in  the  culture. 

5.  Blood-serum. — This  substance,  while  an  important  nutrient  for  a  large  number 
of  bacteria,  is  of  especial  value  for  the  cultivation  of  various  pathogenic  germs  which 
grow  but  feebly  or  not  at  all  upon  the  ordinary  media.     The  serum  is  generally  ob- 
tained from  the  blood  of  beeves  slaughtered  in  the  abattoir,  or  of  the  horses  used  in 
the  places  of  antitoxin  manufacture ;  occasionally  the  blood  of  smaller  animals,  as 
calves,  sheep,  and  the  laboratory  experiment  animals,  is  used.     Exceptionally  the 
serum  is  taken  from  human  blood,  as  that  from  the  placenta.     There  are  differences 
in  the  values  of  the  serum  derived  from  these  various  sources,  but  for  the  ordinary 
scope  of  bacteriologic  work  they  are  not  of  importance.     As  already  suggested,  how- 
ever, this  feature  is  one  to  which  future  study  is  likely  to  add  considerable  interest. 

The  serum  may  be  used  either  in  its  liquid  state  or  after  solidification  by  heat. 
After  coagulation  by  heat  it  cannot  again  be  liquefied,  and  is  therefore  a  suitable  medium 
for  cultivation  at  incubator  temperature.  The  same  fact,  however,  indicates  the 
impossibility  of  this  substance  as  a  medium  for  plate  cultures,  although  mixtures  of 
serum  with  agar  or  gelatine  (from  these,  however,  the  coagulable  albumins  are  lost 
in  the  manufacture)  are  sometimes  employed  for  the  purpose.  Solidified  blood-serum 
is  liquefied  by  the  proteolytic  action  of  bacteria,  but  to  a  less  degree  than  gelatine. 

(a)  The  collection  and  preparation  of  solidified  serum,  especially  by  the  older  method, 
requires  no  little  care  and  time ;  and  the  busy  practitioner  may  without  disadvantage 
for  the  cultivation  of  any  of  the  organisms  requiring  attention  in  clinical  work,  as 
the  diphtheritic  germ,  substitute  for  the  serum  the  whole  blood.  Before  it  has  time 
to  clot  the  blood  is  distributed  to  a  number  of  tubes,  coagulated  by  exposure  to  a 
temperature  of  90°  C.  in  the  oven,  and  then  sterilized  in  the  common  fractional  manner 
in  the  steam-bath.  The  opaque  black  surface  of  the  medium  thus  made  contrasts 
sharply  with  the  pale  or  white  colonies  of  most  of  the  organisms  and  is  not  disad- 
vantageous ;  nor  is  the  cultural  value  of  the  whole  blood  appreciably  less  for  the  majority 
of  bacteria  than  that  of  the  solidified  serum. 

(6)  When  the  serum  is  to  be  used  as  a  liquid  medium  (instead  of  bouillon),  it 


96 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


must  be  kept  in  mind  that  it  is  impossible  to  subject  it  to  temperatures  capable  of 
certainly  sterilizing  it,  and  that  therefore  the  utmost  precautions  must  be  observed 
in  its  collection  and  distribution  to  tubes  to  prevent  contamination.  The  same  care 
is  to  be  exercised  in  the  collection  when  the  older  method  of  solidification  and 
sterilization  is  to  be  pursued,  as  the  clear,  straw-colored  appearance  of  the  product 
cannot  be  preserved  if  it  should  become  necessary  to  sterilize  the  substance  at  a  tem- 
perature above  80°  to  90°  C.  If,  however,  the  white  opaque  solid  (like  boiled  white 
of  egg)  is  intended,  no  particular  precaution  is  essential,  as  this  product  may  be 
sterilized  without  injury  in  steam  in  the  ordinary  fractional  manner.  Even  in  such 
case  some  care  that  no  decomposition  should  have  taken  place  before  preparation 
of  the  medium  should  be  had,  lest  the  natural  faint  alkalinity  of  the  blood  be  altered 
and  other  important  constitutional  changes  have  been  induced.  Usually  no  attempt 
at  adjustment  of  the  reaction  is  made,  the  natural  reaction  of  the  serum  being  suffi- 
ciently constant  and  perhaps  in  some  cases  a  favorable  condition  for  the  growth  of 
bacteria  inoculated  upon  it. 

In  conditions  permitting  care  in  collection  these  details  should  be  followed  in 

order  to  preserve  the  natural  asepsis  of 
the  blood :  A  tall,  narrow  jar,  with  cover 
which  may  be  sealed  against  the  entrance 
of  atmospheric  contamination,  is  sterilized 

TnT  TRT  Tl —  $          *n  a  *ar&e  autoclave,  or  by  boiling ;  or  the 

sterilization  may  be  performed  by  disinfect  - 
ing  solution  (as  of  mercuric  chloride,  or  of 
carbolic  and  hydrochloric  acids),  with  sub- 
sequent rinsing  with  well-boiled  water,  al- 
cohol, and  finally  ether.  It  will  be  found 
convenient  if  the  cover  of  the  jar  has  had 
two  openings  drilled  into  it,  these  being 
closed  after  sterilization  by  sterile  cotton 
plugs.  A  suitable  rubber  tube,  five  or  six 
feet  in  length,  and  of  diameter  easily  enter- 
ing one  of  the  openings  in  the  cover  of  the 
jar  is  similarly  sterilized  and  rinsed  in  well- 
boiled  water  and  wrapped  in  a  sterile  towel 

or  other  protective  until  required  for  use.  In  one  end  of  this  a  glass  canula  should  have 
been  fitted  for  insertion  into  one  of  the  jugular  veins  of  the  animal  from  which  it  is 
expected  to  obtain  the  blood.  At  the  abattoir  the  free  end  of  the  rubber  tube  is  slipped 
into  one  of  the  openings  of  the  cover  of  the  jar  and  fastened  so  as  not  to  be  easily  with- 
drawn, the  end  attached  to  the  canula  being  meanwhile  kept  under  cover.  When 
the  animal  is  felled,  it  should  at  once  be  drawn  up  by  the  hind-legs  until  the  head 
swings  almost  clear  of  the  floor.  The  head  is  then  dragged  to  one  side  and  the  exposed 
side  of  the  neck  cut.  At  once  the  canula,  with  as  little  exposure  as  possible,  is  slipped 
into  one  of  the  cut  veins,  and  the  blood  allowed  to  enter  the  jar.  (If  the  throat  is 
cut  completely,  as  is  usual,  the  operation  becomes  unnecessarily  bloody  and  filthy.) 
As  soon  as  the  jar  is  filled,  the  tube  is  withdrawn  and  the  cotton  stopper  readjusted. 
If  no  more  blood  is  to  be  obtained,  the  tube  is  now  washed  well  in  a  normal  salt  solution 
to  prevent  the  clotting  of  adhering  blood  upon  the  surface  and  in  the  interior.  The 
full  jar  is  set  aside  for  fifteen  or  twenty  minutes  for  the  clot  to  form;  when  this  has 
occurred,  a  long,  stiff  piece  of  wire  is  sterilized  in  the  flame  and  inserted  into  one  of 


FIG.  28. — SERIES  OF  TUBES  ARRANGED  FOR 
COLLECTION  AND  SEDIMENTATION  OF 
BLOOD-SERUM. 


98 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


the  openings  in  the  cover  of  the  jar  and  swept  about  the  sides  of  the  interior  so  as 
to  break  the  clot  away  from  the  glass,  and  thus  permit  its  complete  shrinkage.  The 
wire  is  then  withdrawn  and  the  opening  closed  with  its  cotton  plug.  Thereafter  the 
jar  should  be  transferred  to  the  laboratory  with  as  little  disturbance  of  its  contents 
as  possible  and  placed  in  the  refrigerator  for  twenty-four  to  forty-eight  hours  for 
thorough  separation  of  the  serum  from  the  clot.  In  the  mean  time  a  series  of  large 
test-tubes  or  narrow  flasks  of  suitable  size  are  arranged  for  receiving  the  serum  and 
sedimentation  of  the  blood-cells  which  are  apt  to  be  transferred  with  it  (Fig.  28).  In 
such  a  series  of  sedimentation  tubes  the  connecting  glass  tubes  are  set  into  double- 
perforated  rubber  stoppers.  All  the  parts  are  first  well  cleaned,  the  stoppers  loosely 


FIG.  29. — BLOOD  TUBE  WITH  SIPHON  ARRANGED  TO  TRANSFER  SERUM  FROM  JAR  TO 
SEDIMENTATION  TUBES  ;  TUBE  a  TO  BE  KEPT  ABOVE  LEVEL  OF  SERUM  IN  LAST 
TUBE. 

applied,  and  the  series  folded  together  in  compact  manner  and  sterilized  in  the 
autoclave.  On  removal  from  the  autoclave  the  stoppers  are  at  once  firmly  adjusted 
and  the  series  of  tubes  placed  upon  supports  made  of  blocks  of  wood  with  suitable 
holes  bored  into  them.  A  sterilized  rubber  tube  is  now  inserted  through  one  of  the 
openings  in  the  jar,  well  down  into  the  serum  overlying  the  clot,  and  connected  with 
the  first  tube  of  the  series.  After  this  connection  is  made  a  second  rubber  tube  is 
connected  with  the  escape  of  the  last  of  the  series  to  serve  as  a  mouthpiece,  and  strong 
suction  made  to  start  siphonage  into  the  test-tubes.  If  the  apparatus  has  been  ar- 
ranged with  the  jar  considerably  higher  than  the  series  of  tubes,  the  siphonage 
will  without  difficulty  carry  the  serum  from  one  tube  to  another  in  the  series  and 
nearly  fill  all  (Fig.  29). 


100 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


,1 


The  serum  as  it  passes  into  the  tubes  is  usually  more  or  less  red  and  somewhat 
turbid  from  the  admixture  of  corpuscular  elements  of  the  blood.  The  tubes  are  there- 
fore allowed  to  stand  for  several  hours  longer  in  the  refrigerator,  when  it  will  be  found 
that  the  red  blood  will  have  settled  to  the  bottom  and  the  supernatant,  clear,  straw- 
colored  serum  can  with  little  difficulty  be  forced  from  each  tube  disconnected  from 
the  series,  as  from  a  wash-bottle,  by  placing  a  plug  of  sterile  cotton  in  the  open  end 
of  the  short  tube  and  blowing  through  it.  When  this  is  done,  the  long  end  of  the 
other  tube  should  have  been  slightly  withdrawn  from  the  red  sediment  in  the  bottom 
of  the  sedimentation  tube  lest  this  be  forced  into  the  culture  tubes  (Fig.  30). 

Other  methods  of  distribution  are  often  suggested  or  may  be  devised  as  circum- 
stances demand.  One  of  the  most  simple  and  efficient  is  accomplished  by  means  of  a 
sterilized  pipette  (of  fifty  or  one  hundred  cubic  centimeters  capacity)  having  the 
upper  end  protected  from  the  entrance  of  organisms  by  a  sterile  cotton  plug,  the  serum 

being  drawn  into  such  a  pipette  by  suction  by 
the  mouth  of  the  operator. 

After  distribution  of  the  serum  to  the  cuK 
ture  tubes  (or  dishes,  if  desired)  the  further 
manipulation  consists  in  subjecting  it  to  low 
heating  (Pasteurization — 60°  C.)  for  an  hour 
each  day  for  five  or  six  days,  in  order  to  insure 
the  medium  from  the  influences  of  organisms 
which  have  possibly  gained  entrance  to  it 
during  the  steps  of  collection  or  distribution. 
Upon  the  sixth  or  seventh  day,  with  the  tubes 
adjusted  in  a  slanting  position  to  procure  a 
large  surface  of  exposure,  the  serum  is  gradu- 
ally heated  above  70°  C.  until  it  assumes  the 
appearance  of  a  stiff,  clear,  straw-colored  jelly. 
During  this  stage  the  material  must  be  fre- 
quently examined  lest  it  be  overheated.  Differ- 
ent specimens  coagulate  at  temperatures  vary- 
ing from  70°  to  85°  C.  and  from  half  an  hour 
to  two  hours'  exposure.  Usually,  the  longer 
periods  of  exposure  and  lower  temperatures 
applied  are  followed  by  the  most  sightly  pro- 
duct. Before  the  medium  is  employed  for  any 
important  work,  one  or  two  tubes  should  be 

placed  in  the  incubator  at  body  temperature  for  the  development  of  any  organisms 
which  may  possibly  be  present.  Should  growth  take  place  in  these  tubes,  the  entire 
lot  of  tubes  are  to  be  doubted  and  may  be  either  rejected  or  tried  in  the  incubator,  when 
the  sterile  ones  are  selected ;  or  all  may  be  converted  into  the  opaque  white  serum  by 
heating  them  in  the  oven  to  90°  C.  for  about  ten  or  fifteen  minutes,  after  which  they 
may  with  safety  be  sterilized  in  steam  by  the  fractional  method.  In  Pasteurizing 
and  coagulating  the  serum  in  the  above  manner  it  is  convenient  to  employ  a  special 
warming  chamber,  known  as  a  serum  inspissator,  consisting  of  a  chamber  surrounded 
with  a  water-bath  to  preserve  evenness  and  uniformity  of  temperature,  the  apparatus 
being  provided  with  legs  which  may  be  adjusted  so  as  to  allow  the  tubes  in  the  interior 
to  lie  in  a  slanting  position.  In  such  a  chamber  the  tubes  should  be  placed  upon  a 
layer  of  cotton  or  other  material  to  keep  them  from  direct  contact  with  the  walls, 


FIG.  30. — DISTRIBUTION  FROM  SEDI- 
MENTATION TUBE  TO  CULTURE 
TUBES  ;  TUBULE  a  RAISED  ABOVE 
RED  SEDIMENT  IN  SEDIMENTATION 
TUBE. 


102  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

which  are  somewhat  hotter  than  the  interior  atmosphere;  and  a  thermometer  should 
be  included  with  the  tubes,  which  may  be  watched  through  the  glass  cover  lest  the 
heat  rise  too  high.  A  thermostat  set  several  degrees  higher  than  the  temperature 
desired  in  the  interior  chamber  should  be  placed  in  the  water-bath  of  the  inspissator 
and  connected  with  the  flame  so  as  to  prevent  the  temperature  from  becoming  ex- 
cessive (Fig.  31). 

(c}  When  the  serum  is  to  be  used  as  a  liquid  medium,  the  preparation  is  precisely 
similar  to  the  above,  save  that  the  last  step  of  the  process — that  of  coagulation — 
is  omitted.  The  tubes  in  this  case  also  should  be  tested  for  growth  in  the  incubator. 

(d)  In  recent  years  it  has  become  customary  to  omit  many  of  the  precautionary 
steps  of  the  above  mode  of  operation  and  accept  as  a  satisfactory  product  a  white, 
opaque,  thoroughly  coagulated  serum  susceptible  of  subsequent  sterilization  by  frac- 
tional steaming.  With  such  purpose  in  mind  the  collection  of  the  serum  is  simplified 
to  permitting  it  to  flow  from  the  severed  vessels  of  the  sacrificed  animal  into  the  sterile 
jar,  from  which  the  cover  has  been  completely  removed  for  the  purpose,  only  taking 


FIG.  31. — BLOOD-SERUM  INSPISSATOR,  KOCH  PATTERN. 

the  ordinary  precautions  of  reasonable  cleanliness.  When  the  jar  has  been  filled, 
the  cover  is  readjusted,  and  the  jar  set  aside  until  coagulation  has  taken  place.  The 
cover  is  then  again  removed  and  with  a  sterile  glass  rod  or  wire  the  clot  is  broken 
from  the  sides  of  the  jar.  This  done,  the  cover  is  reapplied  and  the  jar  conveyed 
with  as  little  agitation  as  possible  to  the  laboratory  and  placed  in  the  refrigerator 
The  temperature  of  the  refrigerator  should  not  be  very  low  lest  it  interfere  with  the 
further  clotting  of  the  blood  and  the  efficient  shrinkage  of  the  clot,  but  should  be 
sufficient  to  prevent  the  development  of  the  microorganisms  which  have  probably 
gotten  into  the  blood  in  its  collection.  After  one  or  two  days  in  the  refrigerator  the 
serum  will  be  found  well  separated  from  the  shrunken  clot  and  may  best  be  removed 
by  means  of. sterilized  pipettes.  .It  is  placed  in  a  tall  sedimentation  jar  for  a  few  hours, 
and  after  the  blood  cells  have  settled  to  the  bottom  and  have  left  the  supernatant 
serum  clear,  the  latter  is  transferred  by  means  of  sterile  pipettes  to  sterile  culture 
tubes.  These  are  then  placed  into  the  dry-air  oven,  each  tube  in  a  slanting  position, 
and  heated  cautiously  to  90°  C.,  when  the  serum  will  solidify  into  a  dense,  opaque, 


104  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

white  mass,  like  the  boiled  white  of  an  egg.  This  step  should  not  be  hastened,  and  the 
temperature  should  not  be  allowed  to  go  above  the  limit  mentioned,  lest  the  surface 
of  the  solid  serum  be  blistered  by  bubbles  and  rendered  unfit  for  use.  After  the  serum 
is  thus  solidified  firmly  it  is  transferred  to  the  steam-bath  and  sterilized  in  streaming 
steam  by  the  fractional  method. 

A  fault  in  the  product  of  both  the  old  and  the  present  methods  of  preparation 
is  due  to  the  formation  of  a  dense  film  on  the  surface  of  the  serum,  from  drying,  within 
a  comparatively  short  time.  To  obviate  this  it  may  be  recommended  that  a  drop  of 
glycerine  should  be  placed  in  each  tube  of  the  liquid  serum  before  it  is  subjected  to 
coagulation;  or  it  may  be  prevented  by  covering  the  mouth  of  the  tube  with  a  rubber 
cap  or  similar  device. 

Exercise  29.- — Each  student,  having  previously  prepared  a  pipette 
(fifty  cubic  centimeters)  by  placing  a  cotton  stopper  in  the  upper  end 
and  then  sterilizing  in  the  autoclave,  should  withdraw  fifty  cubic  centi- 
meters of  serum  from  the  sedimentation  jar,  where  it  has  previously  been 
collected  from  the  blood,  before  the  class  and  distribute  it  to  one  dozen 
culture  tubes.  These  are  then  arranged  in  proper  slanting  position  in 
a  wire  cage  and  placed  in  the  dry-air  oven,  where  they  are  to  be  heated 
until  coagulated  firmly.  Heating  should  be  cautiously  done,  the  tem- 
perature raised  slowly  to  8o°-85°  C.  and  maintained  at  this  point  until 
the  coagulation  is  definitely  started,  when  it  is  further  increased  to  90° 
C.  for  ten  minutes  longer.  It  will  probably  require  from  the  first  about 
half  to  three-quarters  of  an  hour  for  completion  of  the  coagulation,  and 
close  watch  should  be  maintained  upon  the  thermometer  of  the  oven 
during  the  time.  Overheating  will  cause  failure  from  blistering  the  sur- 
face of  the  serum,  such  tubes  being  of  no  further  service.  After  the  serum 
is  set  the  tubes  are  to  be  transferred  to  the  steam  sterilizer,  the  top  of 
which  is  loosely  applied,  and  steamed  for  twenty  minutes  on  three  suc- 
cessive days. 

Loeffler's  Glucose-bouillon-serum. — For  its  particular  value  in  the  cultivation  of  the 
organism  of  diphtheria  this  modification  is  suggested  by  Loefner  and  is  of  popular  use. 
Three  parts  of  liquid  serum  are  mixed  with  one  part  of  sterile  glucose  bouillon  (prefer- 
ably, the  latter  should  be  made  from  veal) .  The  subsequent  steps  of  preparation  are 
the  same  as  outlined  for  ordinary  serum. 

6.  Milk. — Aside  from  its  value  as  an  ordinary  culture  medium,  milk  is  made 
use  of  in  bacteriologic  work  in  determination  of  the  production  of  rennet-forming 
ferments  by  bacteria,  as  well  as  in  the  study  of  acid  formation.  The  elements  which 
are  of  principal  nutritive  value  to  the  bacteria  in  milk  are  its  proteids,  milk-sugar,  and 
the  salines.  For  such  use  the  milk  should  be  obtained  as  fresh  as  possible  and 
should  be  freed  from  fat.  This  last  may  be  done  by  allowing  the  cream  to  separate 
spontaneously  from  the  milk  in  a  sterile  jar  in  the  refrigerator  and  then  siphoning  or 
pipetting  the  milk  from  beneath  the  layer  of  cream  into  the  culture  tubes;  or,  as  is 
more  convenient  in  most  laboratories,  fresh  separator  milk  from  the  dairy  may  be 
employed  and  at  once  distributed  to  the  tubes.  The  reaction  of  the  milk  should  first 


106  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

have  been  determined,  although  if  it  prove  not  above  -j-2  it  is  not  customary  to 
make  any  closer  adjustment  to  the  standard;  if,  however,  it  be  more  acid  than  this, 
the  proper  correction  should  have  been  made  before  the  milk  is  distributed  to  the 
tubes.  Having  been  tubed,  it  is  then  sterilized  in  the  steam-bath  at  100°  C.  by  the 
usual  fractional  method. 

Litmus  milk  is  prepared  by  adding  to  the  above  sterilized  tubes,  after  faintly 
alkalinizing  the  milk  with  sodium  hydroxide  solution,  sufficient  sterile  litmus  solution 
to  give  a  distinct  but  pale  blue  tinge  to  the  milk.  If  added  prior  to  the  sterilization,  the 
blue  color  of  the  litmus  milk  is  liable  to  be  changed  to  a  dirty  reddish-brown  color. 
The  preparation  is  used  in  the  study  of  acid  production. 

PRESERVATION    OF    MEDIA    AFTJER    PREPARATION. 

In  order  to  prevent  drying  of  the  media  in  tubes,  which  is  likely  to  produce  a 
tough  scum  upon  the  surface  unfavorable  for  the  development  of  bacteria,  and  may 
cause  much  shrinkage  of  the  mass,  it  is  well  to  close  the  mouths  of  the  tubes  over 
the  cotton  stoppers  with  rubber  caps,  rubber  stoppers,  tinfoil,  or  some  other  device. 
Before  capping  a  tube  the  protruding  portion  of  the  cotton  stopper  should  be 
trimmed  off  with  a  scissors  and  the  surface  of  the  cut  end  of  the  stopper,  as  well  as  the 
lip  of  the  tube,  flamed  to  destroy  any  infection  which  may  possibly  persist.  *  The  cap, 
or  whatever  else  is  employed  as  a  cover,  should  have  been  disinfected  in  a  carbolized 
solution  and  rinsed  in  well-boiled  water  before  application.  Should  these  precautions 
be  omitted  or  the  stopper  not  have  been  originally  thoroughly  sterilized,  the  moisture 
evaporating  from  the  medium  in  the  tube  is  likely  to  collect  in  the  stopper  and  favor 
growth  of  any  infectious  elements  which  may  not  have  been  destroyed.  Properly 
applied,  however,  a  rubber  stopper  or  cap  over  the  cotton  plug  is  of  much  advantage 
in  the  preservation  of  the  medium. 

The  same  end  may  be  attained  by  keeping  the  tubes  of  nutrient  substance  in 
covered  jars ;  these  should  be  well  sterilized  before  the  tubes  are  placed  in  them.  More- 
over, the  tubes  should  be  transferred  to  the  jars  directly  from  the  sterilizer,  before 
mould-spores  or  bacteria  have  chanced  to  come  in  contact  with  the  cotton  stoppers ; 
otherwise  the  moisture  sure  to  be  retained  in  the  cotton  will  favor  the  most  profuse 
growth  of  contaminating  organisms  in  the  plugs  and  these  will  eventually  penetrate  the 
latter  and  gain  entrance  to  the  nutrient  substance  in  the  tubes.  If  a  small  piece  of  gum 
camphor  be  placed  in  such  a  jar,  it  will  be  found  of  considerable  service  in  checking 
the  development  of  moulds,  the  small  amount  of  camphor  absorbed  by  the  medium 
from  the  air  of  the  jar  not  being  of  consequence  usually. 

In  summer  weather,  especially  in  southern  latitudes,  it  is  best  to  keep  gelatine 
tubes,  properly  sealed  and  capped,  in  the  refrigerator,  as  the  prevailing  temperature 
is  usually  sufficient  to  liquefy  the  medium. 


LESSON  V. 

INOCULATION  OF  MEDIA  AND  CULTIVATION  OF 

BACTERIA. 

For  convenience  of  description  it  is  supposed  that  the  student  in  the  work  of  this 
and  the  following  two  lessons  is  dealing  with  material  containing  but  a  single  form  of 
microorganism  and  that  the  cultures  obtained  are  pure,  although- in  practice  this  can 
scarcely  be  expected  to  be  realized. 


APPLIANCES  FOR  AND  METHODS  OF  INOCULATION. 
The  Platinum  Needle. — There  is  no  other  appliance  so  constantly  used  in  bac- 
teriologic  technique  as  the  platinum  needle.     It  is  made  by  fusing  into  one  end  of  a 
glass  rod  the  end  of  a  bit  of  heavy  (about  No.  27)  platinum  wire,  five  or  six  centimeters 


FIG.  32.— STRAIGHT  PLATINUM  WIRE  AND  PLATINUM  WIRE  LOOP. 

in  length.  Platinum  and  glass  are  employed  for  no  other  reason  than  that  they  lend 
themselves  readily  to  sterilization  in  the  flame  without  danger  of  destruction.  Any 
sort  of  wire,  as  iron  or  copper,  with  or  without  a  handle,  may  be  substituted  should 
necessity  demand  such  extemporization.  A  number  of  special  shapes  have  been 
suggested  for  the  needle,  but  with  a  plain,  straight  wire  and  one  twisted  into  a  small 
loop  at  the  end  (known  as  the  "Oese"  or  "loop"),  one  may  proceed  without  difficulty. 
The  loop  is  generally  selected  when  a  liquid  material  containing  microorganisms  is 
to  be  conveyed  to  the  nutrient  medium,  or  when  the  substance  to  be  inoculated  upon 
the  medium  contains  relatively  few  bacteria ;  it  is  used  mainly  for  surface  inoculations 
upon  the  solid  media  and  in  inoculating  liquid  media.  The  plain  needle  is  used  when 
the  matter  to  be  transferred  is  rich  in  bacteria,  as  in  inoculating  from  an  old  culture 
on  solid  medium  to  a  fresh  tube,  and  in  making  stroke  inoculations  and  puncture 
or  stab  inoculations.  If  bent  at  right  angles  to  itself  at  the  very  end  of  the  wire  this 
last  needle  is  especially  suited  for  picking  out  some  special  growth  from  a  mixture 
of  colonies  in  an  impure  culture  in  order  to  transfer  it  to  a  fresh  tube  for  the  production 
of  a  pure  culture.  In  every  use  of  the  needle  (Fig.  32)  it  must  never  be  forgotten 

108 


110  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

that  it  is  absolutely  necessary,  in  order  to  succeed  and  in  order  to  avoid  danger,  that  the 
needle  should  be  thoroughly  sterilized  in  the  flame  (-v.  exercise  6),  both  before  taking  up 
the  infectious  matter  and  after  inoculation  is  completed. 

If  it  is  desired  to  transfer  some  growth  from  one  tube  to  a  tube  of  fresh  solid 
medium,  the  two  tubes  are  grasped  in  the  left  hand  as  shown  in  the  accompanying 
figure  (Fig.  33),  the  upper  ends  of  the  tubes  being  held  well  over  the  ulnar  edge  of  the 
palm.  The  cotton  plugs  may  then  be  removed  with  a  pair  of  dissecting  forceps  and 
laid  upon  a  fresh  piece  of  paper  (or  may  have  been  removed  hy  being  grasped  between 
the  fingers  of  the  left  hand  before  the  tubes  were  placed  in  position,  the  loose  ends 
being  held  between  the  fingers  and  the  deeper  ends  projecting  beyond  the  dorsal  surface 
of  the  fingers).  The  tubes  should  be  held  in  a  horizontal  position  or  slightly  inverted 
so  as  to  prevent  as  much  as  possible  the  entrance  of  chance  organisms  from  the  atmos- 
phere ;  this  position  is  not  possible  if  one  or  other  of  the  tubes  contains  liquid  material, 
in  which  case  the  tubes  must  be  kept  upright.  After  the  stoppers  have  been  disposed 
of  and  the  tubes  properly  held,the  needle  is  taken  in  the  right  hand,  held  as  one  would 


FIG.  33. — PROPER  MODE  OF  HOLDING  TUBES  OF  SOLID  MEDIUM  IN  INOCULATION. 

hold  a  pen,  and  flamed  until  the  whole  length  of  the  wire  has  taken  on  a  cherry-red  color, 
and  the  lower  end  of  the  glass  handle  has  been  exposed  to  the  flame.  For  a  few  moments 
it  is  allowed  to  cool  so  that  its  heat  will  not  prove  fatal  to  the  germs  with  which  it 
'is  to  be  brought  in  contact  (v.  Fig.  2).  As  soon  as  safe  the  needle  is  thrust  into  the 
infected  tube  and  brought  in  light  contact  with  the  culture.  Even  though  one  cannot 
see  it,  many  of  the  individual  germs  adhere  to  the  wire.  It  is  at  once  withdrawn 
and  carried  quickly  and  steadily  into  the  second  tube.  The  needle  may  now  be  drawn 
in  an  even  line  over  the  surface  of  the  medium,  the  growth  which  develops  appearing 
as  a  linear,  more  or  less  continuous  colony,  known  as  a  "stroke"  culture;  or  it  may 
be  rubbed  irregularly  over  the  surface,  when  it  gives  rise  to  an  irregular  film  or  to 
isolated  growths,  which  is  spoken  of  as  a  "smear"  culture;  or  the  plain,  straight  needle 
may  be  thrust  evenly  down  into  the  mass  of  the  medium,  when  the  growth  following 
the  track  of  the  puncture  is  spoken  of  as  a  "puncture"  or  "stab"  culture  (Fig.  34).  The 
loop  is  especially  adapted  for  making  a  smear*  culture.  If  the  medium  to  be  inoculated 
be  liquid,  the  needle  is  merely  agitated  slightly  in  it  in  order  to  set  free  some  of  the 
adherent  organisms  in  the  liquid. 


112 


LABORATORY  EXERCISES  IX  BACTERIOLOGY. 


FIG.  34. — A,  STROKE 
CULTURE.  B. 
PUNCTURE  CUL- 
TURE. 


As  soon  as  the  inoculation  is  accomplished  the  needle  is  to  be  withdrawn  carefully 
from  the  tube  and  flamed  before  being  laid  down;  if  it  be  wet  from  some  liquid  medium 
with  which  it  has  been  brought  in  contact,  it  should  be  held  for 
a  moment  by  the  side  of  the  flame  in  order  to  dry  before  being 
thrust  directly  into  the  flame,  as  the  bubbles  bursting  in  the 
flame  might  scatter  the  infection  and  cause  harm.  The  needle 
having  been  disposed  of,  the  mouths  of  the  two  tubes  are  held 
for  a  moment  in  the  flame  to  destroy  any  infection  which  may 
be  adherent  to  the  lip  and  might  be  forced  into  the  interior  in 
the  application  of  the  stoppers;  and  the  stoppers  are  taken  up 
one  after  the  other  with  a  pair  of  forceps,  flamed,  and  thrust 
into  the  tubes.  (Do  not  mix  the  stoppers.)  The  newly  inocu- 
lated tube  is  then  properly  marked  and  set  aside  for  develop- 
ment. 

The  manipulation  in  using  the  platinum  needle  is  practi- 
cally the  same  as  the  above  from  whatever  source  the  infectious 
material  is  to  be  obtained  or  whether  the  inoculation  is  to  be 
made  upon  media  in  tubes,  dishes,  on  plates,  or  in  flasks.  The 
needle  is  first  sterilized,  then  brought  in  contact  with  the  infec- 
tion, carried  into  contact  with  the  fresh  medium,  and  either 

drawn  over  its  surface  in  a  stroke  or  a  smear  or  thrust  into  the  mass  as  a  stab  (or 
agitated  in  liquid  media),  withdrawn  from  the  medium,  and  flamed;  the  lip  of  the 
tube  or   flask  flamed;  the   stopper  flamed  and   readjusted  (or 
the  cover  of  the  dish  reapplied). 

Swabs. — Sterile  cotton  swabs  are  often  convenient  for  ob- 
taining the  infectious  matter  for  inoculation,  as  from  diph- 
theritic sore  throats  or  the  surface  of  a  wound.  Usually  a 
number  of  such  sterile  swabs  are  kept  ready  in  laboratories, 
inclosed  in  sterilized  test-tubes.  The  tubes  should  be  at  least 
seven  inches  in  length  and  preferably  three-fourths  of  an  inch 
in  diameter,  A  piece  of  ordinary  brass  or  iron  wire  is  cut  a 
little  shorter  than  the  tube  and  one  end  is  roughened  with  a 
file.  About  this  end  a  firm  swab  of  absorbent  cotton  is  twisted, 
and  its  surfaces  rolled  smooth  between  the  fingers.  The  oppo- 
site end  is  set  into  a  small  cork  and  absorbent  cotton  is  firmly 
wrapped  about  it,  completely  covering  in  the  cork,  until  the 
mass  will  fit  closely,  but  not  tightly,  in  the  tube.  The  tube 
having  been  cleaned  in  the  usual  manner,  the  swab  is  intro- 
duced into  its  interior,  the  cotton  plug  wrapped  about  the 
handle  of  the  wire  serving  as  a  stopper  for  the  tube.  The  whole 
appliance  is  now  thoroughly  sterilized  in  the  autoclave  and 
thereafter  a  rubber  cap  is  applied  over  the  mouth  of  the  tube 
to  protect  against  microbic  penetration  and  to  keep  the  swab 
moderately  moist.  The  tube  with  the  contained  swab  may  be 
carried  to  the  bedside,  where  the  swab  is  withdrawn,  brushed 
over  the  infected  surface,  and  at  once  replaced  in  the  tube  and 
carried  to  the  laboratory.  Here  the  swab  is  used  just  as  an 

inoculated  platinum  needle  would  be  used  for  the  making  of  smear  inoculations  upon 
the  surface  of  media  in  tubes  or  dishes  or  on  plates.     After  the  inoculation  has  been 


FIG.  35. — STERILE 
COTTON  SWAB  IN 
TUBE,  WITH  RUB- 
BER CAP  OVER 
MOUTH  OF  LAT- 
TER FOR  GREATER 
PROTECTION  OF 
SWAB  AND  TO 
PREVENT  DRYING. 


114 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


performed,  if  there  be  no  further  need  for  the  material,  the  swab  is  burned  and  the 
tube  disinfected  and  washed  in  the  usual  manner  (Fig.  35). 

Pipettes. — Ordinary  graduated  pipettes  of  small  caliber  are  often  used  for  making 
inoculations  with  definite  quantities  of  liquid  containing  infectious  germs,  and  simple 
pipettes  drawn  in  the  laboratory  from  narrow  glass  tubing  are  likewise  used  for  the 
transfer  of  liquids  to  the  nutrient  media.  The  liquid  thus  added  is  usually  diffused 
through  the  nutrient  which  has  been  previously  liquefied  by  heat,  and  the  contained 
microorganisms  are  thereby  scattered  throughout  the  nutrient  mass.  The  resultant 
culture  thus  grows  either  diffusely  or  in  separate  colonies  (each  representing  at  least 
one  bacterium)  throughout  the  medium;  such  a  culture  is  spoken  of  as  a  "diffusion 
culture."  In  practice,  the  pipette,  the  upper  end  having  been  provided  with  a  cotton 
stopper,  is  sterilized  in  the  usual  manner  in  the  oven  or  autoclave.  If  desired,  a  piece 
of  rubber  tube  may  be  attached  to  the  upper  end  to  serve  as  a  mouthpiece.  The 

small  end  is  passed  several  times  through 
the  flame  as  a  precautionary  measure  and 
then  plunged  into  the  infected  liquid,  which 
is  drawn  into  the  interior  to  the  desired 
graduation  by  suction  by  the  mouth.  A  tube 
of  liquid  medium  is  at  hand  or  a  gelatine  or 
agar  tube  is  liquefied  in  a  water-bath  (either 
one  extemporized  or  of  a  form  especially  de- 
signed for  the  purpose,  as  shown  in  Fig.  36). 
The  stopper  is  removed  from  the  culture 
tube  and  the  measured  quantity  of  the  in- 
fected liquid  passed  into  it.  The  pipette  is 
at  once  laid  aside  (into  a  pan  of  disinfectant 
solution,  if  of  no  more  consequence),  the  lip 
of  the  tube  flamed,  and  the  stopper  flamed 
and  readjusted.  Then,  with  slight  agitation, 
care  being  exercised  that  the  stopper  is  not 
soiled  by  the  contents  of  the  tube,  the 
material  is  thoroughly  diffused  through  the 
medium.  The  tube  may  now  be  set  aside 

and  the  contents  allowed  to  set,  or  the  latter  may  be  poured  into  a  Petri  dish  or 
upon  a  plate  for  a  dish  or  plate  culture,  if  so  desired. 

Should  the  liquid  to  be  inoculated  upon  the  nutrient  medium  contain  but  few 
organisms,  the  usual  amount  employed  is  one  cubic  centimeter,  which  amount  is  gener- 
ally diffused  in  nine  cubic  centimeters  of  the  medium.  If  there  be  numerous  organisms, 
however,  so  that  in  the  resulting  culture  the  colonies  are  likely  to  be  so  numerous 
and  grow  so  close  to  each  other  as  to  interfere  with  proper  observation  and  enumera- 
tion, known  dilutions  with  sterilized  water  may  be  made  to  reduce  the  number  of 
bacteria  in  the  volume  employed — as  one  part  of  the  suspected  fluid  to  nine,  or  ninety- 
nine,  or  nine  hundred  and  ninety-nine  parts  of  sterilized  water.  When  very  small 
fractions  of  a  cubic  centimeter  of  the  original  liquid  or  of  the  known  dilutions  are  de- 
sired, they  are  best  obtained  by  means  of  long  capillary  pipettes  drawn  from  narrow, 
soft  glass  tubing  as  needed.  (To  draw  such  a  pipette  select  an  eight-  or  ten-inch 
piece  of  a  soft  glass  tube  of  small  caliber  and  heat  a  length  of  several  inches  near  the 
middle  in  the  spread  flame  until  it  has  become  ductile ;  then  at  once  remove  from  the 
flame,  and  with  even,  fairly  rapid  traction  from  each  end  draw  the  tube  into  a  uniform 


FIG.  36.  —  LABORATORY  WATER-BATH 
FOR  MELTING  SOLID  MEDIA  IN 
TUBES. 


116  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

capillary  diameter  and  extent  of  several  feet.  As  soon  as  cool  the  tube  is  cut  or  broken 
so  that  as  much  as  possible  of  a  uniform  caliber  of  the  capillary  portion  remains  attached 
to  one  end  of  the  original  glass  tube.)  Into  this  capillary  is  drawn  a  known  quantity 
of  water  (as  0.5  cubic  centimeter)  from  a  watch-crystal  into  wrhich  it  has  been  carefully 
measured.  The  length  of  the  column  of  water  in  the  capillary  is  marked  with  a  pen, 
and  convenient  divisions  for  fractions  of  the  contents  are  similarly  marked  at  proper 
intervals  upon  the  glass.  The  water  is  now  blown  out,  the  tube  washed  with  alcohol 
and  ether  to  insure  dryness,  and  then  sterilized  in  any  large  oven  in  the  usual  manner, 
a  plug  of  cotton  having  been  previously  placed  in  the  larger  end.  (In  the  absence  of 
oven  large  enough  to  accommodate  a  long  pipette,  it  may  be  sterilized  by  drawing  it 
full  of  a  disinfectant  solution  and  immersing  its  capillary  end  in  the  disinfectant,  and 
thereafter  washing  out  the  disinfectant  with  well-boiled  water,  and  rinsing  with  alcohol 
and  ether.)  Thus  prepared  it  is  to  be  used  in  the  same  manner  as  an  ordinary  pipette 
in  inoculation  exercises. 

Syringes. — For  collection  of  liquid  material  and  its  convection  to  the  nutrient 
material  from  a  living  diseased  individual,  as  splenic  blood,  blood  from  a  vein,  the 
contents  of  a  cyst  or  of  an  abscess  or  blister,  syringes  are  often  used.  These  should 
be  of  a  form  and  construction  easy  of  sterilization,  as  Koch's  inoculation  syringe  (Fig. 
37).  All  parts  of  the  syringe  having  been  sterilized  (glass  and  metal  portions  in  auto- 
clave or  oven,  rubber  parts  in  disinfectant  solution  and  boiled  water)  and  adjusted, 


U4 


FIG.  37. — KOCH'S  INOCULATION  SYRINGE. 

the  needle  is  thrust  into  the  tissues  to  the  source  of  the  desired  material  and  the  liquid 
drawn  into  the  syringe.  (Any  part  of  an  exposed  surface  to  be  punctured  should 
have  been  previously  sterilized  in  surgical  fashion.)  Observing  the  usual  precautions, 
the  needle,  after  withdrawal  from  the  tissue,  is  introduced  into  the  culture  tube,  a 
drop  or  more  of  the  contents  expressed  upon  the  nutrient  medium,  the  syringe  disposed 
of,  the  tube  closed  with  the  usual  precautions,  and  the  added  matter  diffused  in  the 
medium  by  gentle  agitation.  The  syringe  is  then  to  be  cleaned  and  sterilized  and  its 
contents  destroyed. 

A  very  convenient  substitute  for  a  syringe  is  the  Sternberg  bulb  (Fig.  38),  which 
may  be  made  in  a  few  minutes  from  a  piece  of  soft  glass  tubing.  (One  end  of  a  piece 
of  glass  tube  about  eight  or  ten  inches  in  length  is  fused  shut  in  the  Bunsen  flame  and 
about  an  inch  of  the  closed  end  softened,  from  which  a  bulb  is  blown  by  the  operator  to 
half  an  inch  or  an  inch  in  diameter.  After  having  become  cool,  the  tube,  as  close  to  the 
bulb  as  possible,  is  softened  in  a  narrow  flame  for  a  distance  of  half  an  inch  or  an  inch 
and  drawn  to  a  fine  capillary  end,  which  if  desired  may  be  sealed  shut  in  the  flame.)  If 
sealed  in  its  manufacture,  the  air  in  the  bulb  will  be  much  rarefied  and  the  interior 
of  the  appliance  may  be  regarded  as  having  been  sterilized  by  the  heat  applied  during 
manufacture.  As  a  uniform  precaution,  however,  it  is  well  to  sterilize  the  bulbs, 
whether  sealed  or  not,  in  a  metal  box  or  wrapped  in  paper,  in  the  oven,  and  the  pro- 
tection of  the  box  or  cover  retained  until  the  bulb  is  to  be  used.  In  use,  the  air  in  the 


118  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

interior  of  the  bulb  is  forced  out  by  holding  it  close  to  a  flame,  and  as  soon  as  this 
is  done  the  capillary  end  of  the  tube  is  plunged  into  the  liquid,  the  latter  being  then 
forced  into  the  interior  by  external  air  pressure.  After  the  liquid  has  entered  the  bulb 
the  capillary  end  may  be  sealed  and  the  bulb  and  its  contents  carried  to  the  laboratory 
for  use  in  inoculation.  (If  the  capillary  end  was  sealed  before  the  collection  of  the 
liquid  it  should  have  been  broken  off  with  a  sterile  pair  of  forceps,  preferably  beneath 
the  surface  of  the  liquid.  If  the  contents  of  a  blister  were  to  have  been  collected, 
the  surface  of  the  lesion  should  first  have  been  sterilized  surgically  and  a  puncture 
made  with  a  sterile  blade  through  which  the  capillary  tube  is  inserted  to  the  interior 
of  the  sac.)  In  the  laboratory  a  tube  of  medium  is  placed  in  a  water-bath  for  lique- 
faction, and  the  capillary  tube  of  the  Sternberg  bulb  disinfected  and  rinsed  in  boiled 
water.  The  plug  is  withdrawn  from  the  culture  tube  in  the  usual  manner,  and  the 
capillary  of  the  bulb  inserted,  the  sealed  end  having  been  broken  off  with  a  sterile 
forceps.  The  close  application  of  the  palm  of  the  hand  to  the  bulb  will  generally 
give  sufficient  warmth  to  force  the  contents  into  the  culture  tube  drop  by  drop;  or  a 
flame  may  be  brought  close  for  the  same  purpose.  After  one  or  more  drops  have  been 
transferred,  the  bulb  is  disposed  of,  the  tube  closed  in  the  usual  fashion,  the  infectious 
material  diffused  through  the  medium  by  agitation,  and  the  tube  set  aside  for  whatever 

purpose  desired.  If  there  be  no  further  need  of  the 
bulb  and  its  contents,  it  should  be  placed  in  a  disin- 
fecting solution  and  subsequently  destroyed  in  the  fur- 
nace. 

Knife-blades. — In  the  course  of  autopsies  or  sur- 
FIG.  38. — STERNBERG  BULB.       gical  operations  scrapings  from  the  tissues  in  suspected 

foci    may    be    transferred   to   the  nutrient  media  by 

means  of  long,  slender,  sterile  knife-blades.  Thus,  if  an  enlarged  gland  is  to  be  sub- 
mitted to  a  culture  examination,  such  a  blade  (sterilized  in  the  autoclave  or  by 
boiling  or  disinfectant  solution — or,  if  not  valuable,  by  flaming)  is  driven  with  its 
cutting  edge  well  into  the  tissue,  turned  on  edge,  and  withdrawn  so  that  the  edge 
will  scrape  some  of  the  pulp  off  the  cut  surface.  A  tube  of  medium  is  taken  up  and 
opened  as  usual,  the  blade  inserted,  and  some  of  the  adherent  pulp  smeared  well 
into  the  surface  of  the  medium  if  solid,  or  the  blade  waved  gently  in  the  medium 
if  the  latter  be  liquid,  so  as  to  dislodge  into  it  some  of  the  pulp.  The  blade  is  then 
withdrawn  and  if  of  no  further  service  is  placed  in  a  dish  of  disinfectant  solution. 
The  lip  of  the  tube  is  flamed,  the  stopper  flamed  and  adjusted,  and  the  tube,  properly 
marked,  is  set  aside  for  development  of  the  inoculated  bacteria. 

Forceps. — Long,  slender,  sterilized  forceps  are  sometimes  used  for  the  convec- 
tion of  infected  solid  substances  to  the  culture  medium. 

Particles. — Under  various  circumstances  small  bits  of  solid  substances  which 
have  been  in  contact  with  the  infectious  matter  may  advantageously  be  themselves 
transferred,  with  the  microorganisms  adhering  to  them,  to  the  nutrient  material. 
For  example,  when  the  upper  end  of  the  culture  tube  has  been  drawn  shut  in  the 
flame,  as  is  occasionally  done  instead  of  closing  with  a  cotton  plug,  it  is  a  matter  of 
some  difficulty  to  arrange  for  the  entrance  of  the  platinum  needle.  In  such  a  case  it 
is  possible  to  take  up  a  bit  of  platinum  wire  in  a  forceps  and  sterilize  it  well  in  the  flame, 
dip  it  into  the  infected  substance,  and  then  drop  it  into  the  tube  through  a  small  open- 
ing made  by  breaking  off  the  tip  of  the  sealed  end.  The  opening  thus  made  is  then 
again  sealed  in  the  flame  and  the  tube  agitated  so  as  to  diffuse  the  bacteria  on  the 
wire  through  the  liquid  medium,  or  over  the  surface  if  the  medium  be  solid.  Some- 


120  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

times,  when  a  comparatively  pure  water  is  to  be  examined,  the  bacteria  from  a  large 
amount  are  concentrated  by  filtration  through  sterile  powder,  and  particles  of  the 
powder  are  transferred  to  the  culture  tube  by  means  of  the  forceps,  carrying  with 
them  a  greater  or  smaller  number  of  the  germs.  Short  lengths  of  capillarv  tube  (sterile), 
into  which  a  suspected  liquid  has  been  drawn,  may  be  broken  off  and  thrown  into 
a  tube  of  liquefied  material  and  the  inclosed  liquid  diffused  through  the  medium  by 
agitation ;  the  known  length  of  the  tube  affording  opportunity  of  approximate  quan- 
titative estimation  of  the  organisms  developing  into  colonies  in  the  medium  if  desired. 
Another  and  somewhat  similar  method  of  approximate  quantitative  estimation  of 
bacteria  in  a  liquid  is  to  take  equal  lengths  of  thread  and  place  one  in  a  liquid  of  the 
same  general  nature  as  that  to  be  examined  but  not  infected  (water  or  bouillon), 
and  a  second  into  the  fluid  to  be  investigated.  The  first  is  weighed  when  wet  and 
placed  in  the  oven  to  dry,  after  which  it  is  again  weighed,  the  difference  in  weight 
representing  the  fluid  it  was  capable  of  absorbing.  The  second  piece  of  thread  is 
taken  from  the  infected  fluid  with  a  sterile  pair  of  forceps,  and  cut  with  sterile  scissors 
at  a  definite  length  (one-half,  third,  or  fourth,  as  desired),  and  the  bit  thus  cut  off 
transferred  to  the  tube  of  liquefied  medium,  and  the  tube  agitated  so  as  to  scatter 
the  bacteria  from  it  throughout  the  medium.  Thereafter  the  tube  is  set  aside  for 
growth  as  a  diffusion  culture  or  its  contents  transferred  to  a  Petri  dish  or  plate 
as  best  suited.  Sterile  granulated  sugar  is  often  used  in  filtration  of  air  and  then 
placed  in  liquefied  medium  so  that  the  sugar  may  dissolve  and  leave  the  bacteria  which 
had  collected  in  the  filter  upon  the  grains  scattered  through  the  medium. 

Fractional  Inoculation. — When  the  substance  to  be  inoculated  into  a  nutrient 
material  is  especially  rich  in  microorganisms,  the  resulting  cultures  are  apt  to  be  so 
crowded  with  colonies,  of  single  or  mixed  type,  that  it  is  impossible  to  recognize  the 
characteristic  appearances  of  a  single  colony  or  to  separate  it  from  the  mass.  To 
prevent  this,  dilution  inoculations  are  generally  made.  A  series  of  three  culture  tubes 
is  usually  used,  the  first  to  be  inoculated  from  the  original  infected  matter,  the  second 
from  the  first  tube,  and  the  third  from  the  second.  Thus,  for  example,  in  the  clinical 
cultivation  of  the  diphtheritic  organism,  Mycobacterium  diphtheria,  three  tubes  of  blood - 
serum  are  usually  inoculated.  The  first  is  inoculated  by  smearing  the  swab,  infected 
from  the  patient's  throat,  over  the  surface  of  the  solidified  serum.  The  platinum  loop 
is  then  taken  up  and  sterilized  and  drawn  over  this  inoculated  surface,  and  then  rubbed 
over  the  surface  of  the  serum  in  the  second  tube.  The  loop  is  again  flamed  and  drawn 
over  the  surface  of  the  medium  in  the  second  tube  and  rubbed  over  that  in  the  third 
tube,  probably  carrying  to  this  last  but  few  of  the  organisms  which  were  originally 
deposited  upon  the  serum  of  the  first  inoculation.  On  comparison  of  the  three  tubes 
after  growth  of  the  infections  the  value  of  the  procedure  will  at  once  be  appreciated, 
the  scattered  colonies  in  the  third  tube  being  much  the  most  easily  studied  and  the 
most  characteristic  in  appearance. 

The  same  principle  is  followed  in  diffusion  inoculations  of  material  rich  in  bacteria. 
The  first  tube  having  been  inoculated  and  the  material  diffused  in  the  liquefied  medium, 
a  given  small  amount  (as  one,  two,  or  three  loopfuls,  or  more  definite  amounts,  as  a 
fraction  of  a  cubic  centimeter,  if  desired)  of  the  mixture  is  carried  into  the  second  tube 
and  similarly  diffused.  The  procedure  is  repeated  from  the  second  to  a  third  tube, 
in  which  the  organisms  of  this  final  dilution  are  likely  to  be  few  and  scattered  and  the 
colonies  resulting  from  their  growth  distinct  and  characteristic. 


122  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

SOURCE  AND  COLLECTION  OF  MATERIAL  FOR  INOCULATION. 

The  wide  range  of  parasitic  and  saprophytic  occurrence  of  bacteria  renders  it 
impossible  to  indicate  any  methodical  procedure  in  their  collection  for  inoculation, 
nearly  every  case  requiring  some  individual  detail  and  particular  precaution  to  insure 
success.  The  physical  character  of  the  substance  to  be  examined  (whether  gaseous, 
liquid,  or  solid),  its  richness  or  poverty  in  bacteria,  the  simplicity  or  complexity  of 
the  bacterial  flora,  and  a  variety  of  other  considerations  all  influence  the  plan  of  pro- 
cedure in  a  greater  or  less  degree. 

In  whatever  manner  the  material  be  obtained  or  whatever  its  source,  as  soon 
as  it  is  removed  from  its  natural  surroundings  and  conditions,  it  is  to  be  kept  until 
actual  inoculation  is  accomplished  in  perfectly  sterile  containers,  contact  with  every 
unsterilized  object  affording  opportunity  for  further  contamination  and  invalidating 
the  results  of  the  study.  So,  too,  the  least  possible  delay  in  inoculation  should  be 
permitted  after  the  acquirement  of  the  infected  material,  especially  if  estimation  of 
the  number  of  bacteria,  as  well  as  of  their  types,  is  to  be  included  in  the  study;  since 
the  chance  for  numerical  increase  or  loss  or  even  of  loss  of  types  in  a  comparatively 
short  period  of  time  after  their  removal  from  their  natural  relations  can  easily  be 
verified.  For  example,  a  water  containing  organic  contamination  may,  owing  to  a 
prevailing  low  temperature,  be  infected  by  comparatively  few  bacteria  when  collected ; 
yet,  if  kept  for  but  a  few  hours  at  the  higher  temperature  of  the  room,  before  inocu- 
lation this  number  may  enormously  multiply  and  the  result  represent  a  condition 
of  contamination  many  times  that  actually  existing  in  the  water  in  nature.  Moreover, 
owing  to  different  rate  of  growth,  one  type  represented  by  few  individuals  in  the  original 
sample  may  in  the  room  temperature  come  to  an  exaggerated  proportion  as  compared 
with  another;  or  the  latter  might  actually  be  crowded  out  of  existence  by  the  an- 
tagonistic influences  of  the  first. 

In  a  general  way  it  may  be  said  that  where  the  infected  material  is  rich  in  bacteria 
and  its  flora  varied  it  is  advisable  to  collect  small  quantities  and  to  further  isolate 
the  bacteria  by  dilution  with  sterile  diluents,  either  before  or  during  the  process  of 
inoculation;  and  that,  on  the  other  hand,  when  a  substance  is  poor  in  its  bacterial 
flora,  in  order  to  more  certainly  obtain  all  the  types  of  its  scattered  organisms  it  is 
essential  that  by  filtration  or  some  other  method  concentration  of  the  organisms  should 
be  accomplished  before  inoculation.  Thus,  sewage  water,  crowded  with  microbic 
life,  must  for  convenience  and  for  reliability  of  results  be  diluted  with  sterile  water 
(or  dilution  inoculation  performed),  often  to  an  extreme  degree;  while  a  pure  potable 
water  should  be  passed  in  large  amounts  through  a  suitable  filter  so  as  to  collect  in 
the  latter  its  occasional,  but  perhaps  variant,  types  in  a  convenient  compass.  The 
organisms  of  the  air  are  likewise  usually  collected  by  filtration  on  sterile  filters.  The 
uncertainty  of  contact  of  the  bacteria  in  a  solid  substance  with  the  nutrient  material 
upon  which  the  solid  may  have  been  inoculated  usually  makes  it  advisable  that  such 
solids  should  as  a  preliminary  measure  be  well  diffused  in  sterile  water,  portions  of 
which  are  thereafter  transferred  to  the  medium,  with  the  result  of  more  certainly 
and  directly  implanting  the  bacteria  upon  the  nutrient. 

In  obtaining  the  infected  material  from  the  living  body,  probably  more  than 
under  any  other  circumstances,  the  necessity  for  precaution  to  prevent  contamination 
by  bacteria  not  concerned  in  the  disease  under  investigation  will  be  appreciated. 
The  presence  of  an  enormous  variety  of  natural  but  unimportant  organisms  in  the 
exposed  parts  of  the  body,  the  difficulties  of  manipulation,  the  uncertainties  in  selective 


124  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

operation,  all  combine  to  render  the  discovery  of  pathogenic  germs  a  most  difficult 
part  of  bacteriologic  investigation. 

Briefly  stated,  it  is  to  be  kept  in  mind,  as  of  fundamental  importance,  that  the 
material  collected  should  be  obtained  so  as  to  represent  its  complete  natural  flora,  that 
it  should  be  preserved  from  contamination  and  intrinsic  changes  before  inoculation  or 
at  once  inoculated  upon  the  nutrient  medium,  and  that  the  infectious  elements  collected 
be  diluted  or  concentrated  by  artificial  device  as  required  for  convenience  of  manipulation 
and  certainty  of  investigation.  For  these  desiderata  the  application  of  method,  care 
of  procedure,  and  a  variety  of  needed  apparatus  must  call  forth  the  ingenuity  and  dex- 
terity of  the  student ;  and  each  case  is  to  be  approached  individually  for  the  application 
of  these  principles,  so  as  to  provide  eventual  success.  It  is  impossible  to  particularly 
describe  the  technique  of  special  procedures,  and  the  consideration  of  a  few  general 
examples,  such  as  will  presently  be  outlined  (air,  water,  milk,  soil,  and  disease  material) , 
must  serve  as  illustration. 

THE  DIFFERENT  FORMS  OF  CULTURES. 

Tube  Cultures.— Growths  of  organisms  inoculated  upon  media  in  test-tubes  are 
least  liable  to  contamination  from  the  various  possible  accidents  of  manipulation, 
and  are  the  most  conveniently  prepared  and  handled;  they  are  therefore  the  type 
usually  employed  unless  some  special  purpose  demands  the  use  of  others.  Cultures 
in  tubes  of  liquid  media  are,  of  course,  diffusion  cultures,  the  organisms  readily  spreading 
through  the  liquid  in  which  they  are  grown.  On  solids,  the  cultures,  from  the  mode 
of  inoculation  and  from  peculiarities  of  the  bacteria  themselves,  may  be  limited  to 
the  surface  (surface  cultures),  as  smear  or  stroke  cultures,  or  may  grow  in  the  interior 
of  the  mass,  as  stab  cultures  or  diffusion  cultures.  The  same  microorganism  grown  by 
these  different  modes  even  upon  the  same  medium  is  likely  to  present  considerable 
differences  of  appearances  and  of  rate  and  profusion  of  development.  Diffusions  and 
smears  usually  show  the  most  characteristic  appearances,  although  both  punctures 
and  stroke  cultures  afford  important  information.  Any  of  the  media  may  be  used 
in  tube  cultures. 

Plate  Cultures. — These  were  introduced  by  Koch  for  use  in  the  separation  of 
mixed  into  pure  cultures,  and  although  largely  superseded  by  Petri  dish  cultures 
and  Esmarch's  tubes,  are  still  frequently  employed.  In  preparing  a  plate  culture, 
the  glass  plates,  supports,  and  culture  dish  already  described  (i>.  Apparatus,  p.  64), 
sterilized  as  indicated,  are  to  be  provided,  three  plates  and  their  platforms  being  usually 
arranged  as  a  set  in  a  culture  dish.  Three  diffusion  inoculations  are  generally  made 
of  the  material  to  be  examined ;  the  first  from  the  original  material,  the  second  by  the 
transfer  of  a  small  amount  (one  or  two  loopfuls)  of  the  first  diffusion  to  the  medium 
in  the  second  tube,  and  the  third  by  like  transfer  from  the  second  to  the  third  tube. 
(Sterilize  the  loop  between  inoculations.)  A  level  surface  is  provided,  usually  by 
means  of  a  platform  tripod  with  adjustable  legs  (Fig.  39),  upon  which  is  placed  a 
flat  dish  filled  level  full  with  ice-water  or  crushed  ice.  This  dish  is  covered  with  a 
large  glass  plate  upon  which  rests  a  glass  bell  jar.  The  glass  plate  and  bell  jar  should 
have  been  disinfected  and  rinsed  in  boiled  water  before  use,  or  otherwise  sterilized. 
One  of  the  plates  intended  for  the  culture  is  placed  upon  the  chilled  surface  of  the 
glass  plate  and  the  liquefied  medium  of  the  first  diffusion  inoculation  poured  over 
it,  the  mouth  of  the  tube  having  been  flamed  before  the  medium  is  poured  out.  The 
bell  jar  is  at  once  lowered,  and  by  tilting  the  glass  cover  of  the  ice  dish  the  liquid  medium 
can  usually  without  difficulty  be  made  to  cover  the  culture  plate  in  a  fairly  even  film. 


126  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

Should  there  be  difficulty,  however,  a  glass  rod  is  quickly  flamed  and  introduced  under 
the  bell  jar  and  used  to  spread  the  medium  before  it  sets.  One  of  the  sterilized  plat- 
forms is  now  placed  in  the  culture  dish  and  the  culture  plate  adjusted  upon  it  as  soon 
as  the  film  of  medium  has  become  solid,  the  cover  of  the  dish  being  immediately 
reapplied.  The  same  process  follows  in  turn  for  the  second  and  third  plates,  upon 
which  are  spread  the  contents  of  the  second  and  third  diffusion  tubes,  the  plates  being, 
arranged  with  their  platforms  in  a  set,  one  over  the  other  in  the  culture  dish.  In 
work  of  this  sort  gelatine  is  usually  used  as  the  medium,  being  more  liquid  at  tem- 
peratures which  are  harmless  to  the  implanted  microbes  than  agar,  which  at  the  same 
temperature  is  less  easily  spread  and  apt  to  quickly  solidify  in  an  uneven  layer  where  it  is 
deposited  on  the  plate.  Gelatine-agar  may,  however,  be  employed,  and  is  preferable 
to  plain  gelatine  in  that  the  dish  and  plates  may  be  introduced  into  the  incubator 
without  danger  of  the  medium  being  liquefied  and  running  off  the  plates. 

Plates  prepared  in  the  same  way  with  uninoculated  medium  are  sometimes  used 
for  smear  or  stroke  inoculation  when  it  is  wished  to  have  a  large  surface  for  distribution 
of  the  infectious  material.  For  this  purpose  well-liquefied  agar  may  be  employed  as 
well  as  gelatine. 


FIG.  39. — LEVELLING  TRIPOD  BEARING  A  DISH  OF  CRACKED  ICE,  COVERED  BY  A  GLASS 
PLATE.  ON  THE  PLATE  A  LEVEL  AND  BELL  JAR  ;  BENEATH  THE  LATTER  A  CULTURE 
PLATE. 

As  a  result  of  spreading  the  diffusely  inoculated  medium  over  the  extensive  surface 
of  the  culture  plate,  the  colonies  arising  from  the  multiplication  of  the  individual 
microbes  are  usually  scattered  and  isolated  from  each  other,  thus  affording  facility 
for  counting  their  number  and  for  examination  of  their  peculiarities  of  form  and  color, 
as  well  as  favoring  any  attempt  at  mechanical  removal  of  one  or  other  colony  to  a 
fresh  medium  in  order  to  obtain  a  pure  culture.  With  a  magnifying  glass  or  the  lowr 
power  of  the  microscope  one  can  frequently  appreciate  peculiarities  of  importance 
in  the  identification  of  the  organisms,  which  might  not  have  been  possible  if  the  colony 
were  in  the  ordinary  culture  tube.  These  advantages  have  made  the  principle  of  plate 
cultures  of  the  utmost  value  to  bacteriologists.  "To  plate  a  tube"  is  an  expression 
often  used  in  the  laboratory,  by  which  is  meant  the  spreading  of  the  contents  of  a 
tube  over  a  broad  surface,  whether  on  plates,  as  above  described,  or  in  Petri  dishes, 
or  over  the  interior  surface  of  the  tube,  as  in  Esmarch  tubes. 

Petri  Dish  Cultures.— The  objectionable  features  of  the  ordinary  plate  culture 
are  the  danger  of  infection  in  preparation  (common  to  all  cultures  in  large  culture 
dishes),  the  rather  tedious  technique  of  spreading  and  solidifying  the  medium  over  a 
cold  surface,  the  possibility  of  the  liquefaction  of  the  medium  during  culture  and 
dripping  from  the  plate,  and  the  difficulty  of  examination  of  the  colonies  without 


128  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

taking  the  plate  out  of  the  culture  dish  and  thus  exposing  it  to  atmospheric  contamina- 
tion. To  offset  these  objections  the  small  flat  dishes  known  as  Petri  dishes  have  been 
introduced.  They  are  used  for  precisely  the  same  purposes  as  the  plates  and  may 
be  thought  of  as  individually  covered  plates.  In  their  employment  the  same  manipu- 
lations are  to  be  practised  as  in  case  of  the  preparation  of  plate  cultures;  but  there 
is  not  the  same  need  of  a  perfectly  level  surface  in  spreading  the  medium  over  the 
bottom  of  such  a  dish,  nor  is  it  requisite  that  the  medium  should  be  immediately 
solidified  by  cold,  as  is  done  in  case  of  plate  cultures,  to  prevent  the  liquid  medium 
from  flowing  over  the  edges  of  the  plate.  The  only  objectionable  feature  in  their 
use — a  very  real  objection,  but  not  nearly  so  serious  as  when  sets  of  plates  are  being 
arranged  in  a  large  culture  dish — is  the  chance  of  entrance  of  contaminating  organisms 
from  the  air  when  the  lid  of  the  dish  is  raised  for  the  introduction  of  the  liquefied 
medium.  From  imperfect  fitting  of  the  lid  to  the  dish,  particularly  in  careless  hand- 
ling, there  is  some  danger  of  entrance  of  bacteria  from  the  outside;  but,  as  already 
suggested,  this  may  be  largely  obviated  by  sealing  the  edge  of  the  cover  over  the 
dish  by  a  strip  of  paper  or  rubber  band  or  other  device. 

Esmarch's  Tubes  (Rolled  Tubes). — As  a  further  development  of  the  same 
principle  Esmarch  suggested  the  absence  of  need  of  exposure  of  the  medium,  either 
in  dish  or  on  plate,  if  the  liquefied  material  after  inoculation  be  spread  over  the  interior 
surface  of  the  tube  itself  and  solidified  in  this  position.  This  idea  leaves  little  to  be 
desired  if  the  amount  of  the  medium  and  the  size  of  the  tube  be  properly  proportioned. 
For  reasons  before  mentioned  gelatine  lends  itself  better  than  the  other  media  for 
the  purpose  involved.  Obviously,  however,  tubes  of  this  form  in  which  gelatine 
is  used  as  the  medium  should  not  be  exposed  to  temperatures  above  25°  to  28°  C., 
lest  the  gelatine  be  liquefied.  Rolled  tubes  of  gelatine-agar  or  of  plain  agar  may, 
however,  be  incubated  without  danger.  In  preparing  a  rolled  tube  a  block  of  ice 
should  be  obtained,  and  its  upper  surface  cut  nearly,  but  not  quite,  level.  A  small 
groove  should  be  cut  in  the  ice,  leading  from  the  higher  edge  over  the  slightly  sloping 
surface  and  its  irregularities  melted  away  by  applying  a  tube  of  hot  water  in  the  groove. 
The  tube  of  liquefied  gelatine  is  now  inoculated,  the  infectious  matter  well  diffused 
by  agitation,  and  it  is  then  laid  in  the  groove  on  the  ice  block,  care  being  taken  that 
the  gelatine  does  not  come  too  close  to  the  stopper.  Here  it  is  steadily  rolled  around 
in  the  groove  until  the  whole  interior  surface  from  the  bottom  to  within  about  half 
an  inch  of  the  stopper  is  evenly  coated  with  a  film  of  the  gelatine  and  the  latter  has 
become  solid.  The  same  end  may  be  accomplished  by  twisting  the  tube  held  in  a 
nearly  horizontal  position  under  a  tap  of  cold  water,  but  the  first  method  is  more 
satisfactory. 

In  such  a  tube  all  the  advantages  of  isolation  of  the  colonies 'for  transfer  to  fresh 
media,  and  for  enumeration  or  examination,  exist  as  in  case  of  plates  or  Petri  dishes, 
and  the  procedure  is  to  be  strongly  commended. 

Flasks. — What  has  been  said  of  tubes  applies  to  flasks,  with  whatever  difference 
the  size  of  the  latter  may  occasion.  The  flask  cultures  are  only  used  when  large  quan- 
tities of  some  isolated  species  are  desired. 


ILLUSTRATIVE  PROCEDURES. 

Examination  of  Air. — The  mere  fact  of  the  presence  of  microbic  life  in  the  atmos- 
phere is  almost  axiomatic,  and  has  been  illustrated  by  the  experiences  gained  in  exercise 
5  or  23,  or  by  the  decomposition  of  media  or  of  organic  substances  used  in  our  daily 


130  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

life  by  infection  of  these  when  accidentally  or  carelessly  exposed  to  the  air.  (In  the 
last  connection,  however,  and  in  the  transmission  of  disease  to  individuals  the  role 
of  flies  and  other  insects  as  conveyers  of  the  infections  must  be  kept  in  mind.)  Both 
vegetative  and  spore  forms  of  bacteria  are  included  in  the  atmospheric  flora.  For 
the  most  part  they  are  attached  to  particles  of  dust  or  of  moisture  in  the  air,  and  for 
this  reason  are  more  numerous  in  the  lower  than  in  the  upper  strata  of  the  atmosphere, 
and  in  dusty  or  moist  air  rather  than  in  a  clean  and  dry  air.  It  is  to  be  presumed 
that  unless  exceptionally  they  do  not  develop  in  the  air,  but  are  conveyed  by  currents 
from  some  point  of  origin ;  for  which  reason  they  are  more  common  in  the  atmosphere 
of  populous  districts,  where  there  is  abundant  opportunity  for  the  development  of 
parasitic  and  saprophytic  germs,  than  in  that  of  dry,  barren,  unpopulated  areas  of 
land  or  in  the  air  over  the  ocean  far  from  the  shore ;  and  for  the  same  reason,  in  the 
atmosphere  of  warm,  moist  localities  and  in  the  summer,  rather  than  in  cold,  dry  climates. 
So,  too,  one  may  expect  to  meet  in  the  air  of  any  selected  locality  more  bacteria  when 
there  are  strong  currents  than  when  it  is  still.  Cornet  has  estimated  the  existence 
of  over  three  hundred  bacteria  in  a  cubic  meter  of  the  air  passing  over  the  house-tops 
of  the  city  of  Paris;  and  thousands  may  be  demonstrated  in  the  same  volume  of  air 
from  the  gutters  and  dirty  cellars  of  cities. 

For  collecting  and  cultivating  these  atmospheric  organisms  a  variety  of  devices 


& 

FIG.  40. 
A.   Sedgwick-Turner  aero-bioscope.     B.  Glass  tube  of  Hesse's  apparatus. 

have  been  suggested,  of  which  the  following  two  may  be  described  as  illustrative  of 
the  principles  of  most : 

Sedgwick-Turner  Aero-bioscope. — This  appliance  (Fig.  40  A),  as  furnished  in  the 
market,  consists  of  a  glass  tube  of  the  shape  shown  in  the  diagram,  and  is  about  thirty- 
five  centimeters  in  total  length.  The  wide  part  of  the  tube  is  fifteen  centimeters  in 
length  and  four  and  a  half  centimeters  in  diameter,  narrowed  at  its  wide  end  to  a 
neck  two  and  a  half  centimeters  in  diameter;  and  at  the  opposite  end  continued  as 
a  narrow  straight  tube  fifteen  centimeters  long  and  one-half  centimeter  in  diameter. 
The  wide  part  of  the  tube  has  squares  marked  upon  its  surface  to  facilitate  counting 
the  colonies  of  bacteria  scattered  over  the  inner  surface  when  the  apparatus  is  in  use. 
A  small  roll  of  wire  gauze  is  placed  in  the  narrow  tube  just  within  the  cotton  stopper, 
extending  to  a  mark  cut  in  the  glass  one-third  of  the  length  of  this  narrow  part  from 
its  free  end.  Both  ends  of  the  appliance  are  plugged  with  cotton  and  the  whole  steril- 
ized in  the  oven  in  the  usual  manner.  Some  finely  granulated  sugar  is  now  introduced 
into  the  wide  end  of  the  tube,  sufficient  to  evenly  fill  the  narrow  tube  above  the  gauze 
on  which  the  sugar  rests.  This  sugar  is  intended  as  a  filter  upon  which  the  bacteria 
of  the  air  will  lodge  and  be  retained  as  air  is  drawn  through  the  apparatus  from  the 


132  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

wide  end  toward  the  narrow  portion.  After  introduction  of  the  sugar  the  cotton 
is  replaced  in  the  wide  mouth  of  the  tube,  the  appliance  held  in  vertical  position  (wide 
end  up)  and  the  sugar  shaken  into  the  narrow  tube.  It  is  now  again  baked  for  steriliza- 
tion of  the  sugar  for  several  hours  at  a  temperature  of  120°  C.  Held  in  the  same 
position,  it  is  then  carried  into  the  atmosphere  to  be  examined,  and  the  lower  end 
attached  to  some  form  of  suction  apparatus,  as  a  pair  of  siphon  bottles  (the  capacity 
of  which  is  known),  the  stopper  removed  from  the  upper  end  of  the  tube,  and  the 
siphonage  started.  After  a  definite  amount  of  air  has  been  drawn  through  the  sugar 
the  cotton  stopper  is  replaced,  the  tube  disconnected  from  the  suction  apparatus, 
and  the  tube  held  in  a  horizontal  position  and  slightly  shaken  to  throw  the  sugar 
back  into  the  wide  part  of  the  tube.  This  done,  twenty-five  cubic  centimeters  of 
liquefied  sterile  gelatine  are  introduced  into  the  expanded  part  of  the  tube  by  means 
of  a  bent  pipette,  the  tube  being  maintained  in  horizontal  position.  In  this  liquid 
the  sugar  is  soon  dissolved,  leaving  the  bacteria  which  were  adherent  scattered  in  the 
medium.  The  medium  is  now  distributed  over  the  inner  surface  of  the  expanded 
part  of  the  apparatus  in  the  same  manner  as  in  any  rolled  tube  and  solidified  by  contact 
with  cold.  It  is  set  aside  at  room  temperature  for  development  of  the  bacteria,  from 
each  of  which  it  is  assumed  a  focus  of  growth  or  colony  will  form  in  due  time.  The 
colonies  are  eventually  counted,  from  the  number  of  which  and  the  known  amount 
of  air  drawn  through  the  tube  the  degree  of  impurity  of  the  atmosphere  may  be  ap- 
preciated. From  the  tube  special  colonies  as  desired  are  to  be  transferred  by  means 
of  the  sterile  platinum  needle  for  further  study  as  isolated  or  pure  cultures,  as  described 
in  a  future  lesson. 

Hesse's  Apparatus. — This  consists  (Fig.  40  B~)  of  a  large,  straight  glass  tube  from 
thirty  to  forty  centimeters  in  length  and  from  four  to  five  centimeters  in  diameter. 
The  ends  of  this  tube  should  be  provided  with  perforated  rubber  stoppers,  into  the 
holes  of  which  are  fitted  short  lengths  of  appropriate  narrow  tube  (in  one  end  the 
tube  fitted  in  the  stopper  should  have  a  comparatively  large  diameter,  1.5  to  2.5 
centimeters,  while  that  of  the  other  end  may  be  about  0.5  centimeter  in  diameter).  The 
large  glass  tube  is  sterilized  in  the  oven  in  the  usual  manner,  the  rubber  stoppers  and 
their  fittings  in  disinfectant  solution  and  rinsed  in  boiled  water.  As  soon  as  ready 
the  stoppers  are  fitted  into  the'tube  and  sterile  cotton  plugs  placed  in  the  small  tubes 
in  the  stoppers.  A  suitable  quantity  of  sterile  gelatine  (twenty-five  to  fifty  cubic 
centimeters)  is  now  liquefied  and  introduced  into  the  tube  by  means  of  a  curved  pipette, 
the  apparatus  being  held  in  a  horizontal  position,  and  distributed  over  the  inner  surface 
and  there  solidified  as  in  the  preparation  of  Esmarch  tubes.  This  done,  the  tube  is 
carried  to  the  atmosphere  to  be  examined,  and  the  narrow  fitted  tube  connected  with 
a  suction  apparatus  (tube  held  in  any  position  after  gelatine  is  solidified),  and  the 
cotton  removed  from  the  other  end.  The  suction  apparatus  is  now  set  in  action  and 
a  definite  quantity  of  air  drawn  through  the  tube.  It  is  supposed  that  the  organisms 
in  the  air,  in  their  progress  from  one  end  to  the  other  of  the  tube,  are  likely  to  come 
in  contact  with  the  gelatine  film  and  adhere  to  it.  The  cotton  stopper  is  then  adjusted, 
the  suction  apparatus  disconnected,  and  the  tube  removed  to  the  laboratory  for  de- 
velopment (at  room  temperature)  of  the  bacteria  which  have  been  introduced.  As 
in  the  method  above  described,  the  resulting  colonies  are  counted,  the  ascertained 
number  being  accepted  as  indicating  at  least  the  same  number  of  individual  bacteria 
to  have  been  present  in  the  amount  of  air  drawn  through  the  tube ;  and  special  colonies 
are  to  be  removed  by  means  of  the  sterilized  platinum  needle  for  isolation  and  further 
study. 


134  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

For  less  exacting  examination  the  mere  exposure  of  sterile  media,  as  potatoes, 
gelatine,  agar,  or  other  substance,  in  open  Petri  dishes  is  usually  sufficient ;  in  such 
case,  however,  one  should  guard  against  the  convection  of  organisms  to  the  nutrient 
substance  by  flies  and  similar  agents. 

Examination  of  Water. — Depending  largely  upon  the  amount  of  organic  im- 
purities (there  are  some  bacteria  capable  of  living  in  filtered  water,  depending  on 
the  minute  impurities  and  carbonic  oxide  absorbed  from  the  air),  the  temperature, 
the  exposure  of  the  water  to  sunlight  (inimical  to  the  bacteria),  and  the  aeration  brought 
about  by  rapid  and  dashing  currents,  and  other  factors,  the  number  and  kind  of 
bacteria  found  in  a  sample  of  water  vary  within  a  wide  range.  Artesian  water,  from 
its  filtration  through  great  distances  of  the  ground,  and  underground  water  generally, 
must  for  mechanical  reasons,  if  for  no  other,  represent  the  purest  types  in  nature. 
No  definite  numerical  limit  may  be  set  for  purity  of  water  from  a  standpoint  of  pota- 
bility, so  many  of  the  common  forms  of  water  bacteria  being  innocuous  when  taken 
in  the  human  system,  and  a  very  few  pathogenic  germs,  on  the  other  hand,  rendering 
a  sample  unfit  for  drinking  purposes.  In  the  most  general  sense,  however,  a  water 
containing  no  more  than  one  hundred  bacteria  to  the  cubic  centimeter  is  regarded 
as  a  pure  water;  above  one  hundred  to  five  hundred  to  the  cubic  centimeter,  as  doubt- 
ful; and  above  five  hundred,  as  unfit  for  drinking  use.  Sewage  water  during  summer 
temperatures  may  sometimes  be  found  to  contain  25,000,000  or  more  organisms  to 
the  cubic  centimeter.  In  studying  any  sample  of  water,  experience  has  shown  that 
the  really  important  bacteria — those  of  diseases  like  typhoid  fever  or  Asiatic  cholera, 
which  are  commonly  believed  to  be  transmitted  through  drinking-water — are  very 
likely  not  to  be  found  even  though  their  presence  is  suspected,  the  conditions  of  collec- 
tion and  cultivation  usually  adopted  not  favoring  their  development  in  the  cultures 
and  consequent  recognition.  They  are  apt  to  be  few  in  the  water  compared  with  the 
ordinary  water  bacteria;  and  the  temperature  of  the  room  usually  employed  for  cul- 
tures and  the  overwhelming  growth  of  the  water  bacteria  probably  interfere  with  their 
best  development.  Should  these  or  other  bacteria,  as  the  colon  bacillus  or  the  putre- 
factive bacteria,  be  recognized,  however,  even  in  the  smallest  numbers,  the  water- 
supply  from  which  they  have  been  obtained  should  without  further  question  be  con- 
demned as  unfit  for  use,  and  corrective  measures  insisted  upon.  Therefore,  in  the 
examination  of  a  sample,  even  though  it  be  impracticable  to  isolate  and  identify  every 
one  of  the  numerous  forms  of  microbes  likely  to  be  encountered,  every  bacteriologic 
analysis  should  occupy  at  least  these  two  phases  of  inquiry :  the  number  of  organisms 
in  a  definite  amount  of  the  sample  (one  cubic  centimeter)  and  the  presence  of  patho- 
genic species  or  such  as  indicate  excrementitious  or  putrefactive  contamination.  More- 
over, such  biologic  examination  should  be  accompanied  by  the  usual  chemical  deter- 
mination of  organic  impurities,  the  number  of  bacteria  becoming  the  more  significant 
as  the  chemical  analysis  indicates  a  small  amount  of  such  contamination.  It  is  not 
the  inert  organic  matter  present  which  is  of  direct  moment,  but  rather  the  active 
life  it  supports  in  the  water ;  not  so  much  the  number  of  these  living  things,  but  essen- 
tially their  character,  which  must  finally  indicate  the  value  or  the  noxious  qualities 
of  the  supply.  As  far  as  the  mere  presence  of  the  water  bacteria  is  concerned,  in 
one  sense  they  must  be  regarded  as  coadjutors  of  man,  in  that  they  destroy  the  organic 
matter  in  water  and  aid  in  its  purification — a  fact  easily  realized  by  one  who  has 
been  accustomed  to  the  use  of  cistern  water  during  warm  seasons.  After  a  rain, 
when  there  are  carried  quantities  of  organic  refuse  from  the  roof  into  the  cistern  from 
rotting  shingles  and  from  the  surface  dust  blown  on  the  roof  by  the  wind,  in  a  favoring 


136  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

temperature  an  active  fermentation  prevails  in  the  water  for  a  number  of  days,  during 
which  time  the  water  is  apt  to  be  turbid  and  foul  and  its  surface  covered  with  froth ; 
in  the  course  of  three  or  four  days,  however,  the  activity  of  the  process  subsides  and 
the  water  again  becomes  clear  and  attractive  to  the  taste,  the  organic  substances 
having  been  destroyed. 

Great  variation  in  the  number  of  bacteria  found  in  a  water  is  certain  to  accompany 
marked  differences  of  temperature;  for  which  reason  it  is  a  fairer  statement  of  the 
condition  of  the  water  to  announce  a  mean  result  from  several  examinations  made  at 
different  seasons,  than  to  give  out  the  result  of  a  single  investigation. 

It  is  essential,  too,  that  the  transfer  of  the  specimen  to  the  culture  medium  should 
follow  its  collection  as  closely  as  possible;  otherwise,  owing  to  the  favor  of  the  quiet 
and  warmth  of  the  laboratory  in  which  it  is  standing,  there  will  be  induced  a  great 
and  rapid  multiplication  of  the  germs  in  the  sample,  vitiating  the  results  of  the  analysis. 
Should  it  be  impossible  to  at  once  proceed  with  the  cultivation  of  the  water  after  it 
has  been  collected,  as  where  it  must  occupy  some  time  in  transit  to  the  laboratory, 
it  should  be  packed  in  ice  to  prevent  this  source  of  error. 

The  method  of  collection  must  vary  with  the  relative  degree  of  bacterial  con- 
tamination. A  small  sample  of  a  water  rich  in  microorganisms  will  in  all  probability 
contain  a  full  representation  of  the  flora  of  the  general  supply ;  when  the  water  is  rela- 
tively pure,  the  same  amount  is  very  likely  to  represent  it  uncertainly  both  numerically 
and  typically.  In  the  latter  instance  it  will  be  advisable  to  practise  some  measure 
which  will  insure  the  concentration  of  the  flora  of  a  large  amount  of  the  water,  as 
upon  some  form  of  sterile  filter,  the  washings  from  which  may  thus  be  taken  as  repre- 
sentative of  the  whole  amount  filtered. 

Water  in  which  currents  prevail  is  likely  to  have  its  bacteria  and  other  particles 
well  disseminated  through  the  whole  bulk,  and  a  single  sample  is  therefore  usually  a 
fair  example  of  the  whole.  When  water  is  stagnant,  however,  the  upper  and  lower 
strata  are  usually  particularly  rich  in  bacteria,  the  former  with  those  in  active  growth 
near  a  free  supply  of  air,  the  latter  from  sedimentation  from  the  higher  strata  and 
with  bacteria  developing  in  the  dead  organic  deposit  and  away  from  a  free  oxygen- 
supply.  In  the  latter  case,  as  in  cisterns  or  infrequently  used  wells,  it  should  be  a 
rule  to  take  samples  from  different  depths, — top,  middle,  and  bottom, — the  report 
of  results  being  the  mean  of  the  series.  So,  too,  when  water  from  a  tap  is  to  be  ex- 
amined, because  of  the  tendency  for  similar  sedimentation  through  the  relatively 
quiet  column  of  water  in  the  ordinary  house  pipes  down  to  the  first  free  current  in 
a  neighboring  main,  it  is  the  usual  practice  to  let  the  water  flow  freely  for  at  least  a 
half  hour  before  the  sample  is  taken. 

It  is  advantageous,  if  there  be  no  reason  to  prevent  and  no  basis  for  reasonable 
opinion  as  to  whether  there  be  large  numbers  or  few  bacteria  in  a  sample  to  be  ex- 
amined, that  a  rough  preliminary  test  be  made  by  inoculating  a  tube  of  liquefied 
gelatine  with  one  cubic  centimeter  of  the  natural  water,  diffusing  and  plating  it.  In 
two  or  three  days  one  can  easily  determine  the  need  for  concentration  of  the  specimen 
or  for  its  dilution  as  there  appear  few  or  many  colonies.  If  it  be  found  that  few  or 
no  growths  follow  this  preliminary  test,  a  filter  is  arranged,  to  be  attached  to  the  tap 
after  the  water  has  been  running  from  it  for  a  proper  time  (or  to  a  large  sterile  funnel 
into  which  the  water  is  poured  if  taken  from  some  source  without  pipe  connection). 
There  is  selected  a  glass  or  tinned  iron  cylinder  about  twenty  or  thirty  centimeters 
in  length  and  one  and  a  half  or  twTo  centimeters  in  the  inside  diameter.  In  the  interior, 
as  shown  in  the  accompanying  diagram  (Fig.  41),  the  folloAving  layers  are  arranged: 


10 


138 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


At  the  lower  end  a  small  roll  of  fine  wire  gauze ;  above  this  a  layer  of  absorbent  cotton ; 
over  the  latter  a  layer  of  fine  sand  about  three  or  four  centimeters  deep;  and  above 
this,  about  the  same  thickness  of  a  fine  powder,  as  chalk,  talcum,  soapstone,  pumice, 
porcelain,  or  glass.  'Thus  arranged,  the  apparatus  is  sterilized  in  the  oven 'by  pro- 
longed baking  and  then  a  small  amount  of  sterile  water  is  poured  into  it  to  moisten 
the  different  layers  and  cause  the  powder  to  become  well  packed.  It  is  now  attached 
to  the  tap  by  a  sterile  rubber  tube  (bound  tightly  with  copper  wire),  the  water  turned 


FIG.  41.—  COLLECTION   FILTER,  FOR  USE  IN   CONCENTRATING  BACTERIA  FROM   A   LARGE 

AMOUNT  OF  WATER. 

A.  Wire  gauze.     B.   Cotton.      C.  Sand.     D.  Powder.     Note   recurved  entrance  tube  at  top 
of  filter  to  prevent  force  of  water  current  from  disturbing  the  powder  in  filter  below. 


on  in  a  gentle  stream  and  a  vessel  of  known  capacity  placed  beneath  by  which  the 
amount  of  water  which  has  passed  through  the  filter  may  be  measured.  After  a 
suitable  quantity  has  been  filtered  (ten  or  twenty  gallons)  the  apparatus  is  detached 
and  the  powder  and  sand  shaken  down  into  a  measured  quantity  (five  hundred  cubic 
centimeters)  and  well  diffused  in  it.  A  known  quantity  of  the  latter,  turbid  with 
grains  of  sand,  powder,  and  bacteria,  is  then  transferred  to  a  tube  of  liquefied  gelatine, 
in  which  it  is  diffused  by  agitation  and  the  medium  thus  inoculated  is  plated  in  one 
of  the  usual  manners.  After  growth  (usually  about  three  days)  at  room  temperature 


140 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


the  developed  colonies  are  counted ;  from  which  number  the  total  in  the  original  quan- 
tity of  the  water  passed  through  the  filter  can  readily  be  calculated.  Special  colonies 
are  removed  from  the  medium,  by  means  of  the  sterile  needle,  for  isolation  and  further 
study. 

If  in  the  test  examination  the  plate  was  crowded  with  growth,  dilution  of  the 
sample  should  be  practised,  sterile  water  being  added  as  the  diluent.  The  sample,  a 
small  one,  is  collected  in  a  sterilized  glass  bottle  provided  with  glass  stopper;  and 
should  it  have  been  standing  for  a  short  time  in  this,  it  should  be  well  shaken  so  as  to 
evenly  distribute  the  organisms  contained,  these  having  tended  to  settle  to  the  bottom. 

Several  dilutions  are  generally  made  (1:10,  1:100, 
1 :  1000).  From  each  of  these  dilutions  one  cubic 
centimeter  is  transferred  to  a  like  number  of  tubes 
of  liquefied  gelatine,  diffused  by  agitation,  and  the 
medium  plated.  As  a  control  a  small  amount  (0.1 
cubic  centimeter)  of  the  original  specimen  is  similarly 
dealt  with.  The  inoculated  preparations  are  then  put 
aside  at  room  temperature  and  the  bacteria  allowed 
to  grow.  At  the  end  of  the  third  or  fourth  day 
development  will  probably  be  complete  (daily  obser- 
vation should,  of  course,  have  been  made),  the  colonies 
counted,  the  number  corrected  for  the  dilution,  and 
the  mean  result  determined. 

In  collecting  samples  from  a  cistern,  pool,  or 
well,  from  which  the  test-specimens  are  to  be  taken 
at  different  depths,  it  is  customary  to  use  glass- 
stoppered  bottles  arranged  in  the  following  manner: 
The  bottle  and  stopper  having  been  sterilized,  the 
operator  carries  it  closed  to  the  place  of  collection, 
where  a  stout  cord  of  sufficient  length  to  reach  the 
depth  desired  is  attached  to  the  stopper  and  a  like 
one  tied  to  the  neck  of  the  bottle;  and  a  convenient 
piece  of  rock  or  other  weight  is  attached  to  the 
bottom  of  the  bottle.  It  is  lowered  to  the  desired 
depth,  the  stopper  withdrawn  by  a  pull  on  the  cord 
attached  to  it,  and  the  bottle  allowed  to  be  filled  with 
the  water.  It  is  now  quickly  pulled  to  the  surface, 
but  steadily  and  without  jerks,  a  little  of  the  con- 
tents poured  over  the  stopper  to  rinse  off  the  water 
of  the  upper  strata  through  which  it  was  drawn,  and 
the  stopper  adjusted.  It  is  probable  that  a  little 

water  from  the  overlying  layers  will  have  entered  the  bottle,  but  this  amount  may  be 
disregarded,  the  full  condition  of  the  bottle  and  the  small  size  of  the  neck  preventing 
much  displacement.  To  prevent  this  source  of  error  special  forms  of  collecting  apparatus 
are  sold  in  which  it  is  possible  by  mechanical  device  to  replace  the  stopper  while  the 
bottle  is  in  the  depths  (Fig.  42).  Samples  are  taken  from  the  top,  middle  depth,  and 
bottom,  and  each  used  in  making  cultures  as  above  described,  dilutions  being  made 
as  above,  if  necessary.  Either  the  mean  of  the  results  obtained  or  a  statement  of 
the  result  for  each  depth  should  be  indicated  in  the  report  of  the  analysis. 

It  is  to  be  remembered  in  all  such  procedures  that  the  number  of  organisms  arrived 


FIG.  42. — BOTTLE  IN  FRAME, 
ARRANGED  FOR  COLLECT- 
ING WATER  FROM  DEFINITE 
DEPTHS  AND  REPLACING 
STOPPER  AFTER  ENTRANCE 
OF  WATER. 


142  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

at  from  counting  the  number  of  colonies  developing  in  the  cultures  probably  represents 
incompletely  the  real  proportion  of  bacteria  in  the  sample.  Each  colony  is  supposed  to 
represent  one  original  bacterium,  but  if  the  diffusion  has  not  been  entirely  successful, 
a  clump  of  organisms  might  well  -have  developed  into  a  single  colony;  moreover,  it 
is  quite  probable  that  not  all  of  the  germs  will  have  developed  on  the  medium  selected 
and  at  the  temperature  to  which  the  cultures  were  exposed.  Close  watch  should 
be  maintained  upon  the  preparations  so  that  all  the  colonies  are  included  in  the  enu- 
meration, a  magnifying  glass  being  used  to  detect  the  smaller  ones.  Where  greater 
exactness  is  demanded  it  is  well  that  not  only  gelatine  preparations  be  made,  but 
that  agar  be  also  inoculated,  plates  of  which,  after  being  grown  for  several  days  in 
the  room  temperature,  are  placed  in  the  incubator  in  order  that  organisms  growing 
only  in  warmth  may  be  afforded  opportunity  for  development.  By  this  measure 
one  may  evade  the  difficulty  which  is  apt  to  arise  from  the  liquefaction  of  the  gelatine 
by  the  bacteria,  a  source  of  much  confusion  in  many  cases.  As  a  rule  the  common 
water  bacteria  develop  best  in  these  cultures  during  the  first  few  days  while  exposed 
to  the  temperature  of  the  room,  the  incubator  preventing  their  free  development. 

Especially  upon  these  agar  preparations  at  incubator  temperature  is  there  proba- 
bility of  appearance  of  colonies  of  such  pathogenic  and  parasitic  organisms  as  may 
have  been  present  in  the  sample.  In  ordinary  investigations  they  are  very  likely 
to  be  overlooked  and  lost.  Of  course,  their  recognition  must  depend  largely  upon 
the  skill,  experience,  and  watchfulness  of  the  observer;  but  in  the  midst  of  the  hundreds 
of  colonies  of  much  the  same  general  appearance  on  the  plate,  even  should  they  appear 
they  may  be  unrecognized  by  the  well-informed  and  skilled.  In  order  to  favor  their 
recognition,  it  is  best  to  induce  their  increase  in  the  sample  of  water  in  some  way 
before  subjecting  it  to  culture,  and  then  make  cultures  on  agar  preparations  at  incu- 
bator temperature.  This  modification  of  the  original  sample  is  not  to  be  made,  of 
course,  until  after  the  preparation  of  the  cultures  above  described,  which  are  intended 
for  the  exhibition  of  the  common  bacteria  and  for  their  enumeration.  Thereafter 
there  may  be  added  to  the  remainder  of  the  sample  in  the  collecting  bottle  either 
peptone  (one  per  cent.)  and  sodium  chloride  (one-half  per  cent.)  or  a  small  amount 
.  of  sterile  bouillon ;  after  which  the  bottle  and  the  contents  are  placed  in  the  incubator 
for  twenty-four  or  forty-eight  hours  for  the  more  satisfactory  development  of  the 
suspected  bacteria.  Under  such  conditions  of  added  nutrition  and  warmth  the  typhoid 
organism,  the  colon  bacillus,  and  other  important  forms  are  apt  to  increase  to  a  marked 
extent,  while  the  common  water  germs  develop  but  poorly  or  are  prevented  entirely. 
Diffusion  or  smear  preparations  on  solid  media  are  now  made  from  this  and  grown 
in  the  incubator.  The  final  recognition  and  identification  of  such  organisms  must 
depend  thereafter  upon  the  experience  and  skill  of  the  observer,  the  separation  of  the 
typhoid  organism  from  the  other  varieties  of  the  colon  group  being  a  difficult  and 
often  uncertain  task,  although  the  recognition  of  the  organisms  as  members  of  this 
group  is  not  difficult  and  quite  convincing  as  evidence  of  the  fecal  contamination 
of  the  sample  of  water  from  which  they  were  obtained. 

Milk  Examination. — Milk  as  obtained  directly  from  the  cow  is  invariably  con- 
taminated with  bacteria  which  have  been  on  the  surface  of  the  teats  or  which  have 
been  growing  in  the  milk  close  to  the  openings  of  the  milk  ducts.  Under  the  most 
rigid  cleanliness  the  fresh  milk  is  apt  to  contain  from  ten  to  twenty  thousand  bacteria 
to  the  cubic  centimeter ;  and  milk  is  often  supplied  to  purchasers  which  contains  as 
many  millions  to  the  same  volume.  The  New  York  Board  of  Health  prescribes  as  a 
limit  for  good  milk  two  hundred  thousand  to  the  cubic  centimeter.  For  the  most 


144  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

part  these  bacteria  in  fresh  milk  are  non-pathogenic  cocci,  although  occasionally  the 
mycobacterium  of  tuberculosis  is  met  with  as  a  serious  contamination;  and  from  air 
or  water  contamination  a  variety  of  more  or  less  serious  infections  may  be  met  with 
in  older  samples.  The  analysis  of  milk  by  culture  follows  the  methods  used  for  water 
examination.  The  milk  is  always  diluted  and  very  small  quantities  of  the  dilute 
sample  (1:  100  or  1:  1000)  employed  for  diffusion  in  the  nutrient  medium,  both  for 
the  purpose  of  reducing  the  number  of  the  bacteria  and  the  confusion  of  their  colonies, 
and  to  prevent  an  annoying  turbidity  in  the  medium  caused  by  the  fat  of  the  milk. 

Soil  Examination. — The  surface  of  the  ground  for  a  variable  depth,  depending 
on  its  porosity  and  the  amount  of  surface  contamination,  is  occupied  by  a  great  variety 
of  organisms,  growing  upon  the  organic  matter  diffused  by  drainage  through  the 
pores  of  the  ground.  These  organisms  ordinarily  are  found  in  profusion  in  the  upper 
three  or  four  feet,  becoming  less  numerous  at  greater  depths ;  and  after  the  intervention 
of  a  bed  of  rock  or  firm  clay  they  are  usually  no  longer  met  with.  For  the  most  part 
they  are  saprophytes  of  little  medical  interest,  concerned  in  the  conversion  of  the 
organic  substances  in  the  ground  into  carbon  dioxide,  water,  and  ammonium;  but 
the  organisms  of  tetanus,  malignant  edema,  black-leg,  and  several  other  affections 
are  also  often  encountered,  and  from  recent  contamination  with  effluvia  from  diseased 


FIG.  43. — HARPOON  FOR  COLLECTING  SOIL  FROM  BELOW  THE  SURFACE  OF  THE  GROUND. 

individuals  the  germs  of  typhoid  fever,  anthrax,  cholera,  and  a  number  of  other  dis- 
eases may  also  temporarily  exist.  One  especially  important  group  of  soil  organisms 
are  the  nitrifying  bacteria,  universally  present,  but  not  capable  of  cultivation  upon 
the  ordinary  media,  and  hence  not  recognized  in  the  usual  bacteriologic  analysis  of 
soil.  They  are  concerned  in  the  oxidation  of  ammoniacal  compounds  in  the  soil  into 
nitrous  and  nitric  acid  and  their  salts,  which  are  then  utilized  by  the  growing  vegetation  ; 
hence  their  importance  to  the  agriculturist  from  their  r61e  in  the  fertilization  of  the 
soil.  They  are  grown  upon  solutions  of  ammonium  sulphate  and  phosphate  of  potas- 
sium, or  upon  a  mixture  of  these  together  with  magnesium  sulphate,  calcium  chloride, 
and  sodium  carbonate  added  to  silicic  acid,  this  mixture  producing  a  mass  of  gelatinous 
consistence. 

In  examining  the  soil,  specimens  are  taken  from  different  depths;  a  convenient 
harpoon  for  obtaining  the  samples  is  that  shown  in  figure  43.  A  definite  amount  of 
the  soil  obtained  is  diffused  in  a  known  quantity  of  sterile  water,  and  a  measured 
quantity  of  this  dilution  with  the  bacteria  scattered  through  it  is  planted  by  diffusion 
in  the  nutrient  medium.  This  is  then  plated  in  one  or  other  of  the  usual  manners 
and  set  aside  for  growth ;  from  the  number  of  resultant  colonies  the  number  of  bacteria 
in  the  original  specimen  is  calculated;  and  the  further  study  of  their  characteristics 
pursued. 

Material   Obtained   from   Diseased   Individuals. — In   the   bacteriologic   study 


146  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

of  the  various  infectious  diseases  the  first  object  of  the  investigator  is  the  disco-very, 
in  the  tissues  or  fluids  of  the  diseased  individual,  of  a  probable  microorganismal  agent. 
For  the  accomplishment  of  this,  the  first  of  the  familiar  postulates  of  Koch,  two  methods 
of  procedure  are  available  and  both  should  be  pursued :  the  examination  of  the  diseased 
material  by  the  microscope  and  the  practice  of  inoculation  of  nutrient  media  with 
such  material.  The  examination  by  the  microscope  may  be  directed  to  the  fresh, 
unstained  matter  or,  better,  to  stained  films  smeared  and  fixed  upon  a  slide  or  cover- 
glass  (see  Smear  Preparations,  Lesson  VII). 

For  successful  culture  of  the  pathogenic  bacteria  blood-serum  probably  offers 
the  greatest  promise  of  any  of  the  common  nutrient  media ;  but,  as  in  any  other  study 
concerned  with  unknown  forms  of  micro-organisms,  one  should  at  least  in  the  pre- 
liminary inoculations  have  recourse  to  the  entire  range  of  laboratory  nutrients,  several 
inoculations  being  made  upon  each  type  to  insure  the  fullest  chance  of  avoiding  failure. 
Here  may  be  recalled  the  method  of  McLaughlin,  originally  devised  and  employed 
by  him  in  the  study  of  dengue  fever,  of  employing  as  the  artificial  nutrient  the  natural 
body  material  supposed  to  be  the  habitat  of  the  organisms  sought.  This  investigator 
withdrew  from  veins  into  sterile  glass  tubes  some  of  the  blood  of  the  infected  subject 
and  with  it  the  organisms  he  has  described,  thereafter  keeping  the  blood  in  the  collection 
tube  in  the  incubator  for  culture  of  the  parasites.  The  method  has  wide  application, 
especially  for  infected  blood  and  fluid  exudates,  excretions  and  secretions,  but  also 
for  infected  solids,  and  has  been  utilized  by  a  number  of  workers  in  a  variety  of  ways. 
Whatever  media  be  employed,  the  various  methods  of  inoculation  possible  for  each 
should  be  practised  and  the  inoculated  preparations  subjected,  as  hereafter  suggested, 
to  body  temperature  in  the  incubator  as  well  as  to  room  temperature,  and  to  anaerobic 
conditions  as  well  as  to  the  ordinary  atmosphere. 

In  case  of  exposed  lesions,  as  the  surface  of  an  ulcer,  or  a  diphtheritic  throat, 
either  in  the  living  or  the  dead  subject,  the  wire  loop  or  the  swab  may  be  well  used 
for  taking  up  the  infected  matter  for  inoculation.  In  obtaining  blood,  it  may  be 
removed  from  one  of  the  large  superficial  veins  of  the  arm  of  the  living  subject  by 
means  of  a  sterile  hypodermic  syringe  or  from  the  pulp  of  the  spleen,  after  thorough 
disinfection  of  the  skin  and,  in  very, cautious  operations,  after  a  small  superficial  open- 
ing has  been  made  through  the  sterilized  skin  by  a  sterile  knife.  Other  fluids,  as  spinal 
fluid,  peritoneal  or  pleural  exudates,  or  the  contents  of  an  abscess,  may  be  obtained 
in  the  same  way.  When  practicable  for  collection,  Sternberg's  bulbs,  capillary  tubes, 
or  pipettes  may  offer  some  advantages  over  syringes  for  subsequent  convection,  inocu- 
lation, or  culture.  From  the  material  thus  procured  smears  and  stabs  are  to  be  made 
in  solid  media  and  diffusions  in  liquefied  agar  and  gelatine,  the  latter  being  subse- 
quently plated.  When  solid  diseased  tissues  are  removed  by  the  surgeon  and  are  to 
be  submitted  to  bacteriologic  examination,  they  should  at  once  be  placed  in  sterile 
jars  and  hermetically  sealed  for  convection  to  the  laboratory.  After  reception  in 
the  laboratory,  as  soon  as  possible  a  knife-blade  should  be  heated  to  red  heat  and 
plunged  into  the  mass,  thus  producing  a  sterile  track  into  the  midst  of  the  tissue  where 
there  cannot  have  taken  place  any  air  contamination  after  removal  from  the  body. 
Along  this  track  a  heavy  platinum  needle,  heated  to  a  red  heat,  is  carried,  plunged 
through  the  eschar  caused  by  the  knife,  and  allowed  to  cool  in  the  interior.  When 
cool,  it  is  pushed  beyond  the  zone  influenced  by  its  own  heat  and  twisted  about  in 
the  structures  until  it  has  in  all  probability  become  infected.  It  is  then  withdrawn 
and  used  in  making  the  usual  smear,  stab,  and  diffusion  inoculations. 

At  autopsies  it  should  be  made  a  rule  to  obtain  the  material  for  inoculation  as 


148  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

soon  as  possible  after  death  in  order  to  prevent  the  appearance  of  the  organisms  of 
putrefaction  as  a  source  of  error  and  confusion;  and  wherever  an  early  operation  is 
impracticable  the  cadaver  should  be  kept  on  ice  or  in  a  refrigerated  room  to  avoid 
the  same  trouble.  Inoculations  should  be  made  from  all  special  points  of  involvement, 
and  from  the  blood  of  the  heart,  spleen,  any  exudates  in  the  tissues  or  body-cavities, 
the  liver  and  the  kidneys  as  a  matter  of  routine.  In  obtaining  the  material  from 
such  sources  the  same  precautions  to  prevent  contamination  should  be  observed  as 
directed  above  when  dealing  with  tissues  removed  surgically,  penetration  into  the 
interior  of  cavities  and  through  the  capsules  of  organs  being  accomplished  by  a  hot 
blade,  and  the  material  removed  by  sterile  syringe,  needle,  or  other  appliance.  In- 
stead of  an  ordinary  platinum  needle  the  heavy  platinum  harpoon  of  Nuttall,  in  a 
metal  or  glass  handle,  is  of  advantage  in  that  it  is  not  easily  bent  in  penetration  into 
firm  tissue. 

After  inoculations  have  been  made  from  the  various  sources  desired,  not  before, 
films  should  be  smeared  upon  glass  covers  or  slides  from  each  examined  part  for  micro- 
scopic study. 

CULTIVATION  OF  INOCULATED  MEDIA. 

The  conditions  for  growth  and  multiplication  of  bacteria  are  the  availability 
of  a  proper  nutrient  and  moisture,  and  the  presence  of  a  suitable  temperature  and  atmos- 
phere. In  addition,  quiet  and  absence  of  extremes  of  light  or  darkness  should  be  included. 
These  conditions  are  sought  to  be  obtained  in  the  measures  taken  for  the  successful 
cultivation  of  the  germs  in  the  laboratory. 

1.  Nutrient. — Sufficient  has  been  said  concerning  this  feature  in  the  consideration 
of  the  various  media  to  indicate  that  the  important  elements  of  nutrition — carbon, 
hydrogen,  oxygen,  and  nitrogen,  as  well  as  traces  of  other  elements  (as  iron,  sulphur, 
phosphorus,  etc.) — are  obtainable  from  the  media  themselves.     There  are  numerous 
organisms  which  grow  upon  much  more  simple  preparations  than  the  ones  described, 
as  upon  solutions  of  salts ;  and  there  are  probably  a  number  for  whose  artificial  culti- 
vation the  media  thus  far  devised  have  not  approached  sufficiently  the  material  afforded 
by  the  living  body.     The  last  are  truly  obligate  parasites.     The  intricate  changes 
which  are  accomplished  by  the  organisms  in  their  appropriation  of  nutrition  and 
growth  in  these  laboratory  cultures  are,   of  course,   but  poorly  understood.     They 
are  mainly  reduction  processes,   oxidations,   and  hydrations. 

2.  Moisture. — The  moisture  present  in  a  medium  is  an  essential  element;  experi- 
ments in  which  it  has  been  purposely  diminished  show  that  its  loss  exerts  a  disturbing 
influence  upon  the  development  of  the  organisms,  growth  being  most  vigorous  when 
seventy  or  more  per  cent,  of  water  is  present  in  the  composition  of  the  medium,  and 
practically  disappearing  when  it  has  been  reduced  as  low  as  forty  per  cent.     Upon 
this  depends  the  success  of  practical  preservation  of  foods  and  other  organic  sub- 
stances by  drying.     The  drying  of.  the  medium  does  not  necessarily  destroy  the  vital 
possibilities  of  the  bacteria  in  the  substance,  even  though  there  be  no  spores  to  account 
for  the  persistence ;  and  after  months  of  drying  it  is  often  possible  to  see  profuse  growth 
appearing  after  the  addition  of  water  or  bouillon  to  a  dried  culture.     In  order  to  pre- 
vent the  loss  of  moisture  from  the  culture  tubes,  particularly  those  in  the  incubator, 
the  warmth  of  which  would  otherwise  much  hasten  desiccation,  a  number  of  devices 
are  occasionally  resorted  to.     The  tubes  may  be  capped  with  rubber  covers  or  closed 
with  rubber  stoppers  (as  for  preservation  of  the  uninoculated  nutrient  media) ;  or 
the  atmosphere  of  the  incubator  (or  of  whatever  other  inclosure  in  which  the  cultures 


150  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

may  be  kept)  may  be  kept  moist  by  placing  a  small  open  dish  of  water  in  the  chamber 
with  the  cultures,  thus  lowering  the  rate  of  evaporation  from  the  inclosed  media. 
In  potato  dishes  the  purpose  of  the  layer  of  moistened  filter  paper  upon  the  bottom 
of  the  dish  is  to  prevent  any  rapid  desiccation  of  the  potato  substance.  The  addition 
of  glycerine  to  agar  or  serum,  aside  from  its  special  nutritive  value,  affords  an  influence 
toward  the  retention  of  the  moisture  of  the  medium. 

3.  Temperature. — The  influence  of  temperature  on  the  growth  of  bacteria  is  an 
important  one,  leading  to  the  recognition  of  three  classes  of  these  organisms,  according 
as  the  temperature  most  favorable  for  their  development  is  low  (room  temperature, 
15°-20°  C.),  about  body  temperature  (35°-38°  C.),  or  high  (50°-55°  C.,  or  above), 
known  respectively  as  psychrophilic,  mesophilic,  and  thermophilic  bacteria.  To  the 
first  belong  many  of  the  saprophytes,  as  most  of  the  common  water  bacteria;  to  the 
second,  most  of  the  bacteria  parasitic  and  pathogenic  to  animal  life;  to  the  third, 
the  putrefactive  bacteria,  many  of  the  bacteria  found  in  the  ground,  and  others.  In 
relation  to  any  one  bacterium  it  is  customary  to  speak  of  that  lowest  temperature 
at  which  it  first  and  feebly  grows  as  its  minimum  temperature;  that  at  which  it  best 
develops,  as  the  optimum  temperature;  and  that  highest  temperature  .at  which  it  con- 
tinues, although  feebly,  as  its  maximum  temperature.  The  entire  range  from  minimum 
to  maximum  for  most  bacteria  will  be  found  to  extend  about  thirty  degrees,  a  few 
having  much  wider  range,  and  a  number,  particularly  pathogenic  varieties,  a  more 
restricted  one.  Some  forms  are  capable  of  development  in  ice  or  snow,  others  at 
the  highest  heat  of  sun  exposure ;  whence  it  will  be  easily  appreciated  that  the  general 
range  for  the  whole  group  must  extend  from  zero  to  as  high  as  60°  or  70°  C.  For  most 
forms  the  optimum  ranges  from  room  temperature  to  body  temperature. 

In  their  artificial  cultivation  the  temperature  sought  to  be  provided  is  the  optimum, 
or  that  most  favoring  their  growth,  particularly  for  those  easily  disturbed  by  varia- 
tions from  this,  as  is  the  case  with  many  of  the  pathogens.  While  slight  differences 
may  not  be  sufficient  to  destroy  the  vitality  of  these  forms,  their  effect  (especially 
if  increased)  is  to  interfere  with  the  rate  of  development  and  often  to  occasion  im- 
portant alterations  in  the  general  functional  activities  of  the  germs.  Sporulation 
may  take  the  place  of  the  ordinary  vegetative  multiplication,  variations  in  the  shape 
and  size  of  the  individual  organisms  may  be  induced,  changes  in  the  degree  of  virulence 
of  the  pathogenic  varieties  may  occur,  as  well  as  other  modifications  of  their  normal 
manifestations.  These  changes  are  included  under  the  term  pleomorphism  of  bacteria, 
which  may  be  induced  by  important  changes  from  any  of  the  normal  conditions  of 
life  as  well  as  by  alteration  of  the  culture  temperature.  Such  pleomorphism  does 
not  involve  in  any  instance  a  change  of  specific  type,  cessation  of  growth  and  of  life 
occurring  rather  than  absolute  loss  of  type.  Such  variations  may,  however,  persist 
through  generations  under  like  modifications  of  life  conditions.  The  anthrax  organism, 
for  example,  is  thus  lowered  in  its  virulence  by  growth  at  a  temperature  of  40°  C. 
(optimum,  37°  C.)  in  the  manufacture  of  the  vaccine  commonly  used  in  the  immuniza- 
tion of  flocks  and  herds  against  the  virulent  disease. 

Usually  two  temperatures  are  available  in  the  bacteriologic  laboratory,  that 
of  the  room  (15°-20°  C.),  and  the  body  temperature  (37°  C.),  which  is  maintained 
by  means  of  an  incubator  and  is  commonly  spoken  of  as  incubator  temperature.  By 
means  of  incubators  any  desired  temperature  may  be  obtained  and  maintained  uniformly ; 
and  for  special  purposes  in  the  larger  laboratories  there  are  usually  provided  incu- 
bators in  which  temperatures  are  maintained  at  ranges  below  (30°  C.)  and  above 
(40°-45°  C.)  that  of  the  generally  used  warm  chamber  (37°  C.).  An  incubator  is  essen- 


152 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


tially  an  inclosure  the  temperature  of  which  is  maintained  at  a  uniform  degree  by  artifice 
of  some  sort.  The  form  most  efficient  and  usually  used  in  the  laboratory  is  a  chamber 
surrounded,  except  on  the  side  of  the  door,  by  a  water-bath  (Fig.  44),  the  source  of 
heat  being  a  small,  constant  flame  adjusted  beneath  the  apparatus.  For  better  preserva- 
tion of  uniformity  there  is  usually  an  incasement  of  asbestos  felt  on  the  exterior,  except 
over  the  bottom  where  the  flame  is  applied.  A  double  door,  the  inner  portion  of  glass 
(for  inspection  of  the  interior  of  the  chamber  without  exposure)  and  the  outer  of  metal 


FIG.  44. — SECTIONAL  VIEW  OF  INCUBATOR. 

A.  Incubator  chamber.  B.  Ventilation  wall.  C.  Water-bath.  D.  Thermometer.  £. 
Thermostat.  F.  Microchemical  burners.  G.  Felt  covering  of  incubator.  H.  Water 
gauge. 


and  felt,  allows  access  to  the  interior;  and  a  small  opening  is  provided  through  the 
top  for  accommodation  of  the  thermometer,  which  extends  into  the  interior.  Several 
openings  at  the  top  lead  into  the  interior  for  ventilation,  and  into  the  water-bath 
for  filling,  cleaning,  and  the  introduction  into  the  warm  water  of  a  temperature  regulator 
(thermostat}.  This  last  device  is  an  appliance  for  the  automatic  regulation  of  the  size 
of  the  flame  beneath  the  incubator,  causing  it  to  decrease  as  the  warmth  of  the  water 
increases,  or  to  increase  as  the  temperature  of  the  water  falls.  There  are  a  number 
of  forms  applicable  to  gas  or  to  lamp  flames,  as  well  as  special  devices  for  the  regu- 


11 


154 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


lation  of  the  heat  obtained  from  electricity  or  from  steam  or  hot -water  pipes;  the 
general  principle  of  all  depending  upon  the  expansion  or  contraction  of  volume  of  some 
solid  or  liquid  upon  the  application  of  heat  or  loss  of  surrounding  temperature.  A 
gas  thermostat,  as  the  cheap,  and  efficient  form  shown  in  figure  45,  may  be  described 
as  illustrative.  The  tube  A,  a  large  test-tube,  is  connected  by  perforated  rubber 
stoppers,  and  the  co-shaped  tube,  B,  with  the  glass  cylinder,  C,  the  arm  of  B  projecting 
some  distance  into  C.  This  cylinder,  C,  is  closed  above  by  a  rubber  stopper  having 
double  perforation.  One  of  the  tubes,  the  feed  tube,  extending  through  this  stopper 
reaches  into  the  arm  of  the  tube  B,  projecting  through  the 
stopper  in  the  bottom  of  C.  Its  lower  end  is  ground  to  a  bevel, 
and  there  is  a  tiny  hole  bored  through  its  wall  a  little  distance 
above  the  beveled  end.  The  other,  the  exhaust  tube,  extends 
but  a  short  distance  into  the  cylinder.  The  test-tube,  A,  is  filled 
with  a  mixture  of  alcohol  and  ether  or  any  other  fluid  having  a 
large  coefficient  of  expansion  by  heat,  the  mixture  extending 
well  into  the  co-tube.  The  dependent  curve  of  the  latter  tube 
contains  mercury,  wrhose  level  joins  the  fluid  in  the  other  arm  and 
serves  to  inclose  it  and  prevent  its  evaporation.  The  beveled 
end  of  the  supply  tube  reaches  to  the  free  surface  of  the  mer- 
cury in  the  co-tube.  The  large  tube  (A)  containing  the  ex- 
pansile fluid  is  now  inserted  into  the  water-bath  of  the  incu- 
bator, the  rest  of  the  apparatus  projecting  into  the  outer 
atmosphere.  The  supply  tube  is  connected  by  means  of  a 
rubber  tube  with  the  gas-pipe  of  the  laboratory,  and  a  tube  leads 
from  the  exhaust  tube  to  the  burner  beneath  the  incubator. 
As  the  water  of  the  bath  rises  in  temperature  its  warmth 
causes  the  fluid  in  A  to  expand ;  this  in  turn  forces  the  level  of 
the  mercury  in  the  co-tube  to  rise  higher  and  higher  about  the 
beveled  end  of  the  supply  tube,  thus  narrowing  more  and  more 
its  orifice  over  the  mercury.  To  offset  entire  closure  of  the 
opening  over  the  mercury,  otherwise  completely  cutting  off  the 
gas-supply  and  extinguishing  the  flame  beneath  the  incubator, 
the  tiny  hole  in  this  tube  has  been  provided.  As  the  orifice 
of  the  supply  tube  becomes  less  and  less  by  the  elevation  of 
the  mercury  column,  the  gas  passing  to  the  flame  is  diminished 
and  the  flame  lowered.  Gradually  the  temperature  of  the  water 
in  the  bath  decreases  and  with  this  the  fluid  in  A  diminishes  in 
volume  and  allows  the  level  of  the  mercury  to  fall  away  from 
the  beveled  end  of  the  supply.  Again  a  greater  volume  of  gas 
passes  to  the  burner,  the  flame  rises,  and  the  temperature  of  the  water  in  the  bath 
increases.  By  a  careful  adjustment,  if  the  pressure  of  the  gas  be  even,  the  tempera- 
ture of  the  bath,  and  consequently  of  the  incubator  chamber,  may  be  maintained 
indefinitely  within  a  variation  of  less  than  one  degree.  For  delicacy  of  the  appa- 
ratus the  volume  of  the  expansile  fluid  in  A  should  be  large  in  proportion  to  the 
volume  of  mercury  which  it  is  made  to  lift  in  the  co-tube. 

One  of  the  most  annoying  interferences  with  the  maintenance  of  an  even  tem- 
perature arises  from  inequalities  in  the  pressure  of  the  gas  in  the  general  supply  pipes. 
To  obviate  this  the  pipe  connected  with  the  incubator  should  be  connected  as  directly 
as  possible  with  the  main  (near  the  meter)  and  not  attached  to  a  series  having  many 


FIG.   45. — THERMO- 
STAT. 

A.    Tube   containing 
expansile       liquid. 

B.  co-shaped  tube 
containing  in  shad- 
ed   part    mercury. 

C.  Cylinder    serv- 
ing   as   gas  cham- 
ber. D.  Feed  tube. 
E.   Escape  tube. 


156 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


side  connections.  The  variations  in  pressure  in  the  mains  leading  directly  from  the 
gas  works  are  usually  gradual  and  a  good  thermostat  is  capable  of  close  regulation 
from  such  a  source  without  the  need  for  further  intervention ;  but  to  prevent  the  sudden 
changes  in  a  house  pipe  carrying  a  large  number  of  burners  in  frequent  and  intermittent 
use,  a  gas-pressure  regulator  should  be  interposed  between  the  thermostat  and  the 
pipe  from  which  the  incubator  is  supplied.  A  number  of  devices  are  used  for  this 
purpose,  but  the  adaptation  of  the  principle  of  an  ordinary  gas  tank  having  a  constant 
weight  superimposed,  as  in  the  Murrell  regulator  (Fig.  46),  is  probably  the  most  efficient 
form. 

The  cost  of  the  bacteriologic  incubators,  as  well  as  of  other  set  apparatus  used 
in  well-equipped  laboratories,  is  often  prohibitive  to  private  individuals,  who  in  the 
course  of  their  medical  practice  might  otherwise  be  anxious  and  able  to  conduct  in- 
vestigations in  clinical  bacteriology  to  the  advantage  of  themselves  and  their  patients, 
and  to  that  of  science  in  general.  In  this  connection  it  may  therefore  be  suggested 
that  the  ordinary  egg  incubator,  with  a  lamp  as  the  source  of  heat  and  the  thermostat 

used  in  such  an  appliance,  may  easily 
be  arranged  to  answer  every  probable 
demand.  There  is  shown  in  the  ac- 
companying diagram  the  plan  of  a 
cheap  incubator  of  large  size  used 
by  the  writer  for  class  work,  which 
can  be  duplicated  for  a  compara- 
tively small  sum  (Fig.  47).  The 
arrangement  of  the  hot-water  tank 
on  the  inside  of  the  incubator  cham- 
ber, instead  of  as  an  outside  jacket, 
is  not  so  efficient  as  the  latter  in  the 
maintenance  of  an  even  temperature 
through  the  whole  inclosure,  but  it  is 
a  much  cheaper  mode  of  application. 
It  has  been  possible  to  keep  the  tem- 
perature of  selected  parts  of  the  ap- 
FIG.  46.— MURRELL  GAS  PRESSURE  REGULATOR,  paratus  uniform  within  two  degrees, 

and    if   a    proper   circulation  of   the 

heated  water  were  arranged  by  a  series  of  tubes  this  variation  could  be  reduced.  The 
degree  of  warmth  for  each  part  of  the  chamber  is,  however,  constant,  the  warmest 
part  being  in  the  lower  tier  of  wire  cages  near  the  tank,  and  the  coolest  near  the  top 
and  front.  Every  incubator  should  occupy  a  place  in  which  as  few  atmospheric  draughts 
as  possible  prevail,  to  escape  disturbances  of  the  flame  and  to  lessen  the  loss  of  heat  by 
convection.  It  is  well  to  have  a  small  room,  separate  from  the  general  laboratory, 
devoted  to  the  accommodation  of  the  incubators  and  arranged  so  as  to  best  protect 
them. 

As  an  extemporization  in  clinical  work  it  may  be  possible  to  utilize  some  nook 
close  to  a  range  or  house  furnace  for  the  accommodation  of  culture  tubes;  but  this  is 
at  best  precarious.  One  or  two  tubes  of  infected  medium  in  a  small  metal  box,  wrapped 
in  several  layers  of  cloth  or  paper,  and  put  in  an  inner  pocket,  carried  to  bed  at  night, 
may  be  successfully  incubated  by  body  warmth  should  necessity  place  one  in  extremes. 
4.  Atmosphere. — From  their  relations  to  the  oxygen  of  air  bacteria  are  usually 
classified  in  three  groups — obligate  anaerobic  bacteria  (obligate  anaerobes'),  obligate 


158 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


aerobic  bacteria  (obligate  aerobes),  and  facultative  anaerobic  bacteria  (facultative  anaerobes'). 
The  first  group  are  unable  to  vegetate  or  sporulate  except  in  the  absence  of  free  oxygen ; 
the  second,  except  in  the  presence  of  free  oxygen ;  the  third  group,  while  usually  develop- 
ing in  the  presence  of  the  atmospheric  oxygen,  are  able  to  maintain  their  activities 
in  its  absence.  Probably  this  division  should  not  be  accepted  in  an  absolute  sense, 
since  in  case  of  many  of  the  known  aerobes  there  is  the  power  of  adaptation  to  atmos- 
pheres containing  less  and  less  oxygen,  and  the  same  is  true  of  many  anaerobes  grown 
in  atmospheres  of  nitrogen  or  hydrogen  to  which  are  gradually  added  small  proportions 
of  oxygen.  It  has  been  noted,  too,  that  when  living  in  a  culture  with  aerobic  bacteria 
it  is  sometimes  possible  that  development  of  associated  anaerobes  will  occur  in  the 
ordinary  atmosphere,  as  seen  clinically  in  the  association  of  the  bacillus  of  tetanus 
with  the  pyogenic  cocci  in  superficial  wounds.  So,  too,  it  has  been  noted  that  the 


FIG.  47.  LARGE  CLASS   INCUBATOR    WITH    INTERIOR   WARM-WATER    TANK,  AND   WIRE- 
CAGE  DRAWERS,  WALL  OF  WOOD,  COVERED  WITH  FELT. 

A. — Side   section.     /.  Water-bath.     2.   Wire-cage    drawers.       ?.    Partition    between    rows   of 

drawers,  perforated  for  proper  circulation  of  warm  air. 

B. — Front  view,  open  door.      /.   Water-bath.     2  and  j.   Wire-cage  drawers ;  front  of  partitions 
removed  above  to  show  insertion  of  (4}  thermometer  and  (5)  thermostat. 


addition  of  certain  substances,  as  sodium  sulphide  (one  or  two  drops  of  a  ten  per  cent, 
solution  to  ten  cubic  centimeters) ,  to  ordinary  bouillon  or  gelatine  will  enable  many 
anaerobes  to  develop  in  such  media  in  the  ordinary  atmosphere.  On  the  other  hand, 
an  excessive  proportion  of  oxygen  in  the  atmospheres  of  cultures  of  aerobes  may 
modify  their  vital  phenomena  (as  in  the  reduction  of  the  virulence  of  Bacillus  anthracis 
by  excess  of  oxygen)  or  eventually  destroy  them. 

Among  the  anaerobic  organisms  are  many  of  the  soil  bacteria,  that  of  lockjaw- 
being  of  much  pathologic  importance;  among  the  aerobes  are  most  of  the  common 
saprophytes,  while  in  the  group  of  the  facultative  anaerobes  bacteria  pathogenic  and 
parasitic  to  animal  life  are  found. 

Atmospheres  of  nitrogen  or  hydrogen  are  well  suited  for  the  growrth  of  anaerobic 


160 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


bacteria,  and  it  is  said  that  hydrogen  sulphide  is  likewise  favorable;  carbon  dioxide 
is  restrictive.  Mere  reduction  of  atmospheric  pressure  by  the  air-pump  may  be  em- 
ployed for  the  purpose,  but  an  actual  vacuum  is  not  conducive  to  their  development. 
It  is  probable  that,  like  other  living  things,  these  anaerobes  actually  require  oxygen 
for  their  energies,  but  are  able  to  obtain  the  small  amounts  needed  from  the  material 
upon  which  they  are  grown. 

Various  devices  and  special  measures  have  been  suggested  for  the  development 
of  anaerobic  organisms.  In  many  of  these,  jars  of  some  form  or  other  are  prepared 
so  that  an  atmosphere  of  nitrogen  or  hydrogen  may  be  introduced  to  replace  the  ordinary 
air.  A  simple  type  of  such  jar  is  shown  in  Fig.  48.  The  jar,  cover,  and  rubber  ring 
should  first  be  disinfected  and  rinsed  in  boiled  water.  The  inoculated  tube  cultures 
(without  cap  or  rubber  stopper),  small  plate  cultures,  or  open  Petri  dishes  of  small 
size,  are  placed  in  the  jar,  the  cover  adjusted  (taking  care  that  the  long  supply  tube 
extending  from  the  cover  does  not  come  in  contact  with  plate  or  dish  media)  and 
fastened  hermetically.  Hydrogen  gas  is  generated  in  a  Kip  generator  (Fig.  49)  or 
other  apparatus  from  dilute  sulphuric  acid  and  zinc  clippings  (gas  preferably  washed 
by  being  passed  through  one  or  two  Woulff  bottles  containing  water),  and  passed 

into  the  supply  tube,  the  clamps  of  both  the  supply 
and  the  escape  tubes  being  loose.  The  hydrogen 
entering  the  lower  part  of  the  jar  gradually  dis- 
places the  air  which  passes  out  through  the  exit  tube. 
It  is  possible  to  determine  the  purity  of  the  hydrogen 
gas  after  it  has  passed  through  the  jar  (air  expelled) 
by  collecting  some  of  the  escaping  gas  under  water  in  ' 
a  test-tube  and  applying  a  flame.  If  no  explosion 
follows,  the  air  has  all  been  expelled.  However,  if 
one  will  allow  the  gas  to  flow  freely  for  about  five 
minutes,  then  close  the  clamps  for  a  time,  and  after 
five  or  ten  minutes  more  (allowed  for  diffusion  of 
gas  in  the  tubes  of  the  cultures)  repeat  the  operation, 
he  may  be  confident  of  having  obtained  the  desired 
condition.  The  Hoffmann  clamps  are  then  tight- 
ened, and  the  jar  placed  in  the  proper  temperature  for  the  best  development  of  the 
organisms. 

Another  simple  method  is  to  rely  on  the  removal  of  the  oxygen  from  the  atmosphere 
by  means  of  an  alkalinized  solution  of  pyrogallic  acid.  Buchner  suggests  that  a  large 
test-tube  be  employed,  the  inoculated  culture  tube  being  placed  in  the  interior.  A 
rubber  stopper  is  obtained  by  which  to  seal  the  large  tube.  Several  cubic  centimeters 
of  a  saturated  solution  of  pyrogallic  acid  are  introduced  into  the  large  tube  outside 
the  culture  tube,  and  alkalinized  by  adding  an  equal  amount  of  a  strong  (forty  per 
cent.)  solution  of  sodium  hydroxide,  and  the  rubber  stopper  at  once  applied.  The 
oxygen  is  absorbed  by  the  mixture,  leaving  an  atmosphere  mainly  composed  of  nitrogen. 
The  writer  has  for  a  number  of  years  employed  with  some  success  the  measure 
for  excluding  the  atmosphere  from  the  tube  cultures  by  filling  the  tube  after  a  stab 
or  diffusion  inoculation  has  been  made  with  the  same  medium  as  that  in  the  tube  or 
with  paraffine  still  liquid,  but  near  the  temperature  for  solidification.  Sterile  oil  may 
be  poured  over  the  inoculated  medium  for  the  same  purpose.  On  plates  a  sheet  of 
sterile  isinglass  or  thin  glass  closely  applied  to  the  surface  of  the  medium  may  suffi- 
ciently exclude  the  air  from  the  underlying  medium  to  be  followed  by  growth  of  anae- 


FIG.  48. — ANAEROBIC  JAR. 


162 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


robic  varieties.  Salomonsen's  capillary  tubes  drawn  full  of  the  inoculated  medium, 
and  the  ends  sealed,  will  often  show  marked  growths  of  the  anaerobes;  and  if  in  the 
fermentation  tube  growth  be  limited  to  the  closed  arm  of  the  apparatus,  it  may  be 
inferred  that  the  organism  present  is  capable  of  anaerobic  development. 

5.  Light. — Direct  sunlight  is  destructive  to  most  organisms,  even  diffuse  light 
if  moderately  strong  being  restrictive  to  many.  On  the  other  hand,  absolute  and 
constant  darkness  is  not  favorable  for  their  best  development,  the  most  suitable  con- 


FIG.  49. — KIP  GAS  GENERATOR  AND  WASH-BOTTLE. 

dition  being  an  extremely  weak,  diffused  light  alternating  with  periods  of  darkness. 
Cultures  in  the  incubator  are  thus  properly  placed ;  and  it  should  be  a  rule  that  cultures 
growing  at  room  temperature  be  placed  in  a  rather  dark  nook  for  their  best  growth. 


THE  NUMERICAL  ESTIMATION  OF  BACTERIA. 

Sufficient  reference  has  been  made  in  connection  with  the  above  instructions  as 
to  the  mode  of  analysis  of  air,  water,  milk,  and  soil  to  have  the  student  understand 
that  for  this  purpose  diffusion  inoculations  of  known  small  quantities  of  the  original 
infected  material  or  of  known  dilutions  of  such  matter  in  sterile  diluents  are  first  to 
be  made.  As  suggested,  it  is  best  to  make  a  series  of  such  dilutions,  diffusing  each 
in  the  medium  so  as  to  have  a  series  of  cultures  for  comparison  of  results.  After 
diffusion  in  the  medium  (usually  liquefied  gelatine)  in  a  culture  tube,  the  mixed  con- 
tents are  to  be  plated,  either  in  plates,  in  Petri  dishes,  or  in  the  interior  of  the  tube 
(Esmarch  tube),  the  inoculated  bacteria  being  thus  disseminated  over  the  surface 
upon  which  the  film  of  medium  is  distributed.  It  is  to  be  presumed  that  upon  placing 
these  plated  cultures  in  proper  temperature,  atmosphere,  and  in  a  dark  place,  each 
bacterium  will  grow  into  a  colony  more  or  less  separated  from  its  fellows.  The  time 
permitted  for  growth  must  vary  somewhat  with  circumstances,  but  forty-eight  or 


164  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

seventy-two  hours  are  usually  sufficient  for  the  appearance  of  the  colonies  so  that 
they  may  be  detected.  Should  gelatine  have  been  selected  as  the  medium  of  growth, 
the  confusion  arising  from  liquefaction  of  the  medium  may  require  earlier  counting, 
or  that  recourse  to  agar  preparations  as  media  be  had. 

Should  the  number  of  colonies  present  in  the  dish,  plate,  or  tube  be  small,  there 
can,  of  course,  be  no  difficulty  in  their  enumeration ;  but  one  must  always  be  careful 
not  to  overlook  small  colonies,  and  a  magnifying  glass  should  be  used  for  their  detec- 
tion, and  in  order  not  to  mistake  bubbles  or  small  dirt  particles  for  colonies.  If  the 
preparation  be  placed  over  a  dark  surface,  as  black  paper  or  cloth,  the  colonies  stand 
out  more  clearly  and  are  more  easily  recognized. 

If,  however,  there  be  great  numbers  of  bacteria  present,  recourse  must  be  had 
to  some  device  to  aid  the  worker.  Thus,  a  piece  of  black  paper  may  be  ruled  with 
white  lines  into  squares  having  each  side  one  centimeter  in  length,  and  this  then 
placed  beneath  a  plate  or  dish  culture,  through  which  the  lines  are  visible,  dividing  the 
culture  into  square  centimeters.  One  may,  if  time  and  inclination  agree,  proceed 
to  determine  the  number  of  colonies  in  each  of  these  squares  in  regular  system  until 


FIG.  50. — MODE  OF  COUNTING  COLONIES  IN  AN  ESMARCH  TUBE. 

all  have  been  included  in  the  count.  This  is  the  safest  procedure  if  there  be  not  too 
many  colonies.  Or,  having  counted  the  colonies  in  a  number  of  squares  in  different 
parts  of  the  culture,  a  mean  may  be  struck  for  one  square  centimeter.  The  square 
surface  of  the  culture  being  known,  the  calculation  for  the  whole  culture  is  easily 
made.  For  counting  the  number  of  colonies  in  a  Petri  dish  a  black  surface,  marked 
off  by  concentric  circles,  radial  lines,  and  subdivisions,  into  divisions  each  one  square 
centimeter  in  area,  may  be  found  more  convenient  than  the  squarely  ruled  surface. 
In  counting  the  colonies  in  a  rolled  tube  a  strip  of  paper,  in  which  an  opening  of  one 
square  centimeter  size  has  been  cut,  may  be  folded  about  the  tube  (Fig.  50) ;  the  colonies 
showing  in  the  opening  counted,  and  the  slip  removed  from  place  to  place  until  ten 
or  twelve  separate  square  centimeters  have  been  gone  over.  From  the  inner  diameter 
of  the  tube  may  be  calculated  the  circumferential  measure  of  the  film  (diameter  multi- 
plied by  3.14159) ;  this  is  multiplied  by  the  extent  of  the  film  in  the  long  axis  of  the 
tube  (exclusive  of  the  curve  of  the  bottom,  which  may  be  neglected  to  make  correction 
for  the  error  arising  from  the  fact  that  the  inner  circumference  of  the  film  is  less  than 
that  of  the  tube)  and  thus  the  square  surface  of  the  film  obtained.  The  number  of 


166  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

colonies  calculated  for  one  square  centimeter,  multiplied  by  the  number  of .  square 
centimeters  in  the  film,  furnishes  the  total  number  of  bacteria  in  the  material  inocu- 
lated. Or,  if  one  wishes,  the  outer  surface  of  the  tube  may  be  laid  off  in  squares  by 
longitudinal  and  circumferential  lines,  a  wax  pencil  being  used  to  write  on  the  glass, 
these  serving  as  the  squares  laid  beneath  an  ordinary  plate  or  dish  culture.  After 
a  proper  count  of  the  colonies  in  each  culture  of  the  several  dilution  inoculations  has 
been  made,  and  this  number  multiplied  by  the  coefficient  of  dilution  of  the  original 
substance,  the  mean  of  the  results  should  be  taken  as  most  nearly  representing  the 
correct  number  sought.  However,  it  should  be  recalled  that  the  figures  obtained  are 
really  only  approximations,  and  are  understatements  rather  than  exaggerations,  owing 
to  the  failure  of  this  or  that  bacterium  to  develop  into  a  recognizable  colony  for  some 
reason  or  other,  and  owing  to  the  fact  that  some  colonies  may  represent,  not  isolated 
organisms,  but  clumps  of  germs. 

Exercise  30. — Under  the  eyes  of  the  instructor  let  each  student  in  turn 
make  an  inoculation  of  tubes  of  solid  medium  by  stroke  (using  plain 
needle),  smear  (using  loop),  and  stab  inoculations;  and  of  liquid  medium 
by  diffusion  inoculation  (using  loop) .  In  this  it  should  be  seen  that  the 
student  properly  holds  the  tubes  from  which  material  is  obtained  and  to 
which  inoculation  is  to  be  made ;  that  he  removes  the  stoppers  correctly 
and  completely,  and  disposes  of  them  properly;  that  he  flames  the  needle 
properly  both  before  and  after  inoculation,  flames  the  mouth  of  tube  and 
stopper  before  readjusting  the  latter,  and  that  the  whole  process  is  carried 
out  without  undue  exposure  to  contaminations. 

Exercise  31. — Each  student  prepare  a  swab,  sterilizing  it  for  use  in  ob- 
taining material  from  the  throat  of  a  diphtheritic  patient. 

Exercise  32. — Prepare  from  soft  glass  tubing  three  capillary  pipettes,  the 
capillary  portion  of  each  of  which  shall  be  uniform  for  at  least  twelve 
inches;  graduate  each  into  tenths  of  a  cubic  centimeter,  properly  prepar- 
ing it  for  sterilization  in  the  oven  (rinsing  in  alcohol  and  ether,  and 
plugging  end  with  cotton) ;  thereafter  sterilize  in  oven  for  future  exercise. 

Exercise  33. — Each  student  prepare  one  Sternberg  bulb,  at  least  one- 
half  inch  in  diameter  of  bulb  and  having  a  capillary  tube  of  at  least  two 
inches  in  length. 

Exercise  34. — By  means  of  sterile  pipette  measure  one  cubic  centimeter 
of  milk  and  add  it  to  nine  cubic  centimeters  of  sterile  distilled  water  in  a 
small  sterilized  flask,  mixing  by  gentle  agitation.  From  the  mixture,  by 
means  of  a  sterilized  capillary  pipette,  remove  o.i  cubic  centimeter  and 
transfer  to  a  tube  of  liquefied  gelatine  (kept  liquid  in  warm  water  at  30°  C.). 
At  once  diffuse,  and,  following  instructions  given  above,  distribute  the  diffu- 
sion upon  a  sterile  glass  plate  and  place  it  in  a  culture  dish  for  development. 
Remove  from  the  remainder  of  the  decimal  dilution,  by  means  of  the  sterile 
pipette,  one  cubic  centimeter  of  the  dilute  milk ;  with  assistance  of  a  fellow 


168  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

student  at  once  rinse  out  the  flask  with  sterile  distilled  water,  placing 
thereafter  in  it  nine  cubic  centimeters  of  sterile  distilled  water.  To  this 
add  the  one  cubic  centimeter  of  the  decimal  dilution,  thus  preparing  a  one 
per  cent,  dilution,  diffusing  thoroughly  by  agitation.  From  this  second 
dilution  remove  with  sterilized  capillary  pipette  o.  i  cubic  centimeter  and 
transfer  to  a  tube  of  liquefied  agar  (kept  liquid  in  water -bath  at  45°  C.) ; 
diffuse  by  agitation,  and  transfer  the  contents  to  a  Petri  dish,  in  which  it 
should  be  quickly  cooled  so  as  to  harm  as  little  as  possible  the  bacteria  by 
the  temperature  of  the  agar,  placing  the  dish  after  solidification  in  the  in- 
cubator. From  the  one  per  cent,  dilution  remove  with  a  sterile  pipette 
one  cubic  centimeter  of  the  dilute  milk;  again  quickly  rinse  out  the  flask 
with  sterile  distilled  water,  and  place  nine  cubic  centimeters  of  the  sterile 
distilled  water  in  the  flask.  Add  the  one  per  cent,  dilution  to  the  latter, 
thereby  making  a  one  in  a  thousand  dilution.  Of  this,  with  sterile  capillary 
pipette  inoculate  o.  i  cubic  centimeter  to  a  second  tube  of  liquefied  gelatine 
and  roll  over  ice  to  make  an  Ksmarch  tube.  Put  aside  in  a  dark  place  at 
room  temperature  for  further  development.  Observe  each  culture  at 
close  of  the  first,  second,  and  third  days,  and  at  the  close  of  the  last  period 
count  the  number  of  bacteria  obtained  in  each  dilution.  Calculate  there- 
from the  number  in  one  cubic  centimeter  of  the  undiluted  milk.  (Should 
liquefaction  of  medium  endanger  the  definition  of  the  colonies  before  the 
seventy-second  hour  shall  have  been  passed,  count  at  an  earlier  period  as 
conditions  require.) 

Note  the  differences  shown  by  the  colonies  on  the  agar  in  the  incubator 
and  those  of  the  gelatine  plate  and  tube  at  room  temperature. 

(As  far  as  possible  let  the  instructor  witness  the  various  steps  taken  by  each 
student  in  the  processes  of  dilution,  inoculation,  and  the  different  modes  of  plating 
the  inoculated  media,  to  insure  all  necessary  precautions  against  contamination  and 
to  correct  errors  of  manipulation.) 

Exercise  35. — Make  an  inoculation  of  one  cubic  centimeter  of  water 
from  the  tap  or  from  a  cistern  into  a  tube  of  liquefied  agar ;  diffuse  by  agita- 
tion; transfer  to  a  Petri  dish;  grow  in  incubator.  Make  a  similar  inocula- 
tion in  a  tube  of  liquefied  gelatine ;  transfer  to  Petri  dish ;  place  in  a  dark 
place  at  room  temperature  for  development.  Compare  the  rate  of  devel- 
opment of  these  psychrophilic  bacteria  at  the  two  temperatures.  Are 
those  on  the  agar  different  in  gross  appearance  of  colonies  from  those  on  the 
gelatine  ? 

Exercise  36. — Inoculate  two  tubes  of  solid  blood-serum  by  smears  from 
a  known  culture  of  Mycobacterium  diphtheria.  Let  one  remain  at  room 
temperature;  grow  the  second  in  the  incubator  at  body  temperature. 


12 


170  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

Note  difference  in  the  cultures  at  close  of  first,  second,  and  third  days.  To 
what  group  is  the  organism  to  be  referred  from  its  optimum  temperature  ? 

Exercise  37. — From  a  known  culture  of  Bacillus  tetani  a  stab  culture  in 
agar  is  made  (glucose  agar  if  preferred).  It  is  placed  for  forty-eight  hours 
in  the  incubator.  Does  development  occur? 

It  is  now  transferred  to  anaerobic  jar  in  an  atmosphere  of  hydrogen  and 
again  placed  in  the  incubator,  and  observed  for  the  usual  time.  To  what 
class  of  bacteria  does  this  germ  belong  from  its  relations  to  free  air  access  ? 

Exercise  38. — Two  tubes  of  agar  are  inoculated  by  stroke  from  a  known 
culture  of  Bacillus  typhosus.  One  is  placed  in  the  anaerobic  jar  in  an  at- 
mosphere of  hydrogen,  the  other  retained  in  the  air.  Both  are  placed  in 
the  incubator.  Note  the  results  of  growth  at  the  close  of  the  first,  second, 
and  third  days.  To  what  group  does  this  organism  belong  from  its  relation 
to  air? 

Exercise  39. — From  a  known  culture  of  Pseudomonas  pyocyanea  (Bacil- 
lus pyocyaneus)  make  a  stroke  inoculation  on  a  tube  of  agar.  Place  in  an 
atmosphere  of  hydrogen  for  two  days  at  incubator  temperature.  Does 
growth  occur?  Transfer  the  tube  to  the  open  air  in  the  incubator.  Ob- 
serve the  results  after  one,  two,  and  three  days.  Is  this  organism  an 
obligate  anaerobe,  facultative  anaerobe,  or  obligate  aerobe? 

Exercise  40. — One  or  two  loopfuls  of  fresh  pus  are  diffused  in  a  few  cubic 
centimeters  of  sterile  water.  Smear  preparations  are  made  on  two  agar 
'  'slants"  (tubes  in  which  the  medium  has  been  solidified  in  slanting  position). 
One  is  placed  in  the  sunlight  near  the  window,  the  other  in  a  dark  place  at 
the  temperature  of  the  room.  Compare  the  two  tubes  each  day  for  three 
days  and  note  results.  Can  the  culture  exposed  to  sunlight  be  developed 
by  transfer  to  darkness  and  warmth,  or  are  its  organisms  dead? 


LESSON  VI. 

STUDY    OF    GROSS    APPEARANCES    OF    BACTERIAL 

CULTURES. 

Upon  development  of  bacteria  inoculated  upon  a  nutrient  medium  there  appear 
as  a  result  of  multiplication  of  the  individual  organisms  more  or  less  isolated  colonies. 
A  colony  is  a  focus  of  growth,  or  group  of  bacteria,  recognizable  by  the  eye,  unaided, 
or  aided  by  low  powers  of  the  microscope.  Should  such  a  colony,  as  is  usually  the 
case,  be  composed  of  bacteria  of  the  same  type,  arising  from  the  multiplication  of  a 
single  original  germ,  it  may  be  spoken  of  as  a  pure  colony;  while  if  there  be  confused 
in  the  group  several  types,  resulting  from  the  merging  together  of  minute  foci  of  de- 
velopment of  closely  situated  but  different  germs,  it  may  be  termed  an  impure  or 
mixed  colony.  In  liquid  media  the  chances  for  diffusion  of  the  organisms  by  agitation, 
extension  of  growth,  or  motility  of  the  germs  are  so  marked  that  isolation  of  the  colonies 
and  preservation  of  their  separate  characters  are  exceptional;  while  upon  solid  media 
each  separate  focus  of  growth  is  apt  to  maintain  its  isolation  and  peculiarities,  whence 
important  data  for  the  identification  of  the  type  may  be  obtained.  The  study  of 
isolated  colonies  is  naturally  most  readily  pursued  in  diffusion  cultures  on  solid  mediaf 
either  in  the  films  of  plates,  dishes,  or  rolled  tubes,  or  throughout  the  mass  of  the 
medium,  or  in  dilute  smear  cultures  where  the  original  germs  have  been  well  scattered 
over  the  surface.  The  massive  growths  of  stroke  and  stab  inoculations  on  solids  and 
the  unrestrained  diffuse  growths  in  liquid  media  present  less  characteristic  appear- 
ances, but  may  in  individual  cases  contribute  important  information  for  the  same  end. 

An  organism  does  not  preserve  an  identity  of  appearance  of  its  colonies  and  massive 
growths  upon  the  various  media,  or  when  influenced  by  modifications  of  its  other 
life  conditions;  and  the  variations  produced  by  development  upon  different  media 
and  in  different  atmospheres  and  at  different  temperatures  are  likewise  of  importance 
in  identification  of  similar  but  unlike  types  from  each  other.  With  a  view  of  estab- 
lishing these  points  of  peculiarity  it  is  customary,  therefore,  to  study  the  appearances 
of  growth  of  a  given  bacterium  upon  various  media  (potato,  gelatine,  agar,  serum, 
bouillon,  and  milk),  in  each  instance  noting  the  characteristics  shown  by  the  different 
forms  of  inoculation,  as  well  as  the  influences  exerted  by  variations  of  temperature 
and  of  atmosphere  upon  the  development  in  each.  For  complete  study  of  all  its 
peculiarities  the  organism  should  have  been  grown  in  agar  by  smear,  stroke,  stab, 
and  diffusion  (plate  and  in  the  mass  in  a  tube) ;  similarly  in  gelatine ;  by  smear  and 
stroke  inoculation  on  potato  and  blood-serum ;  and  by  diffusion  in  bouillon  and  milk. 
Preparations  of  each  of  these  should  be  submitted  to  at  least  two  temperatures — 
that  of  the  room  and  that  of  the  body;  and,  similarly,  examples  of  each  should  be 
placed  in  anaerobic  conditions  as  well  as  in  the  ordinary  air. 

In  every  investigation  the  rate  of  growth  (time  of  appearance  of  visible  growth), 
optimum  temperature,  preference  of  nutrient  medium  and  of  atmosphere  should  be  first 
noticed  and  recorded.  Thereafter  the  appearances  of  the  cultures  in  each  medium  are 

172 


174  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

in  turn  noted,  both  for  the  isolated  colonies  and  the  diffuse  growths,  including  in  each 
instance  the  position  of  growth  (whether  upon  the  surface  or  beneath  the  surface  of 
the  medium),  the  differences  apparent  in  surface  and  deep  growths,  the  profuseness  or 
poverty  of  growth,  the  color,  shape,  size,  appearance  of  the  margin,  consistence,  optical 
characteristics,  and  internal  structure  of  the  focus  of  growth.  The  elevation  or  depression 
of  the  surface  growths,  liquefaction,  change  of  reaction,  and  other  alterations  of  the  nutrient 
medium,  the  appearance  of  gas  bubbles  in  the  medium  and  any  peculiar  odor  are  re- 
corded. In  liquid  media  are  noted  any  changes  in  transparency  and  color,  the  appear- 
ance of  pellicle  or  sediment;  and  any  changes  in  the  general  appearances  of  the  medium 
(as  coagulation  of  milk)  are  to  be  further  observed.  Modifications  resulting  from 
age  of  culture  are  likewise  to  be  included  in  the  study. 

From  the  fact  that  no  definite  nomenclature  has  been  adopted  in  this,  as  in  other 
branches  of  physical  science,  for  the  description  of  cultural  peculiarities,  and  in  the 
absence  of  a  definite  system  of  observation,  considerable  difficulty  of  recognition  has 
arisen;  and  it  is  often  almost  impossible,  from  the  writings  of  observers  working  with 
the  same  organism,  to  be  sure  of  the  identity  of  their  material.  The  personal  equation 
in  the  description  of  the  form,  color,  and  other  characters  of  a  colony,  the  failure  to 
indicate  as  the  basis  of  description  the  exact  conditions  of  growth,  and  the  loose  use 
of  descriptive  terms  are  largely  the  cause  of  this  confusion;  and  every  effort  to  fix  a 
system  of  observation  and  to  establish  a  well-defined  nomenclature  must  be  com- 
mendable. With  this  purpose  in  view  the  following  list  of  descriptive  terms,  by  permis- 
sion of  the  author,  is  included  in  these  pages,  adopted  from  Chester's  Manual  of  Deter- 
minative Bacteriology. 

(For  discussion  of  color,  gas  formation,  liquefaction  of  medium,  reaction  changes, 
etc.,  see  next  chapter.) 

CHARACTERS  OF  BACTERIAL  CULTURES. 

I.  Gelatine  Stab  Cultures: 

(A)  Non-liquefying: 
Line  of  Puncture: 

Filiform,  uniform  growth,  without  special  characters  (Fig.  51,  1  B). 
Nodose,  consisting  of  closely  aggregated  colonies. 

Beaded,  consisting  of  loosely  placed  or  disjointed  colonies  (Fig.  51,  2  B). 
Papillate,  beset  with  papillate  extensions. 
Echinate,  beset  with  acicular  extensions  (Fig.  51,  3  B). 
Villous,  beset  with  short,  undivided,  hair-like  extensions  (Fig.  51,  5  B). 
Plumose,  a  delicate  feathery  growth. 

Arborescent,   branched  or  tree-like,   beset  with  branched,   hair-like  extensions 
(Fig.  51,  4  B). 

(B)  Liquefying: 

Crateriform,  a  saucer-shaped  liquefaction  of  the  gelatine  (Fig.  52,  1). 
Saccate,  shape  of  an  elongated  sack,  tubular,  cylindric  (Fig.  52,  3). 
Infundibuliform,  shape  of  a  funnel,  conical  (Fig.  52,  4). 
Napiform,  shape  of  a  turnip  (Fig.  52,  2). 

Fusiform,  outline  of  a  parsnip,  narrow  at  either  end,  broadest  below  the  surface. 
Stratiform,  liquefaction    extending  to  the  walls  of  the  tube  and    downward 
horizontally  (Fig.  52,  5). 

II.  Stroke  Culture  (see  plate  cultural  characters). 

III.  Plate  Cultures,  Colonies: 


176 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


(A)   Form : 

Punctiform,   dimensions  too  slight  for  defining  form   by  naked  eye;  minute, 

raised,  semi-spherical. 
Round,  of  a  more  or  less  circular  outline. 
Irregular. 
Elliptical. 

Fusiform,  spindle-shaped,  tapering  at  each  end. 
Cochleate,  spiral  or  twisted  like  a  snail-shell  (Fig.  53,  A). 
Ameboid,  very  irregular,  streaming  (Fig.  53,  B). 


FIG.  51. — GROSS  CULTURAL  APPEARANCES,  NON-LIQUEFYING. — (After  Chester. 

A. — Types  of  elevation  of  cultures.  /.  Flat.  2.  Raised,  j.  Convex.  4.  Pulvinate.  5. 
Capitate.  6.  Umbilicate.  7.  Umbonate. 

B. — Types  of  growth  along  puncture.  /.  Filiform.  2.  Beaded,  j.  Echinate.  4.  Arbores- 
cent j".  Villous. 


Mycelioid,  a  filamentous  colony,  with  the  radiate  character  of  a  mould  (Fig. 

54,  D). 

Filamentous,  an  irregular  mass  of  loosely  woven  filaments  (Fig.  54,  E). 

Floccose,  of  a  dense  wooly  structure. 

Rhizoid,   of  an  irregular  branched,   root-like   character,   as  in   Bact.   mycoides 

(Fig.  53,  C). 

Conglomerate,  an  aggregate  of  colonies  of  similar  size  and  form  (Fig.  55,  A). 
Toruloid,  an  aggregate  of  colonies,  like  the  budding  of  the  yeast  plant  (Fig. 

55,  B). 

Rosufate,  shaped  like  a  rosette. 


178 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


(B)  Surface  Elevation: 

1.  General  Character  of  Surface  as  a  Whole: 

Flat,  thin,  leafy,  spreading  over  the  surface  (Fig.  51,  A   1). 

Effused,  spread  over  the  surface  as  a  thin  veilly  layer,  more  delicate  than  the 

preceding. 

Raised,  growth  thick,  with  abrupt,  terraced  edges  (Fig.  51,  A  2). 
Convex,  surface  the  segment  of  a  circle,  but  very  flatly  convex  (Fig.  51,  A  3). 
Pulvinate,  surface  the  segment  of  a  circle,  but  decidedly  convex  (Fig.  51,  A  4) 
Capitate,  surface  hemispherical  (Fig.  51,  A  5). 

2.  Detailed  Characters  of  Surface: 

Smooth,  surface  even,  without  any  of  the  following  distinctive  characters. 
Alveolate,  marked  by  depressions  separated  by  thin  walls,  so  as  to  resemble  a 
honeycomb  (Fig.  55,  C). 


\J 

FIG.  52. — TYPES  OF  LIQUEFYING  CULTURES. — (After  Chester.} 
i.  Crateriforra.     2.  Napiform.    j.  Saccate.     4.   Infundibuliform.     5.   Stratiform. 

Punctate,  dotted  with  punctures  like  pin-pricks. 

Bullate,  like  a  blistered  surface,  rising  in  convex  prominences,  rather  coarse. 

Vesicular,  more  or  less  covered  with  minute  vesicles  due  to  gas  formation  more 

minute  than  bullate. 

Verrucose,  wart-like,  bearing  wart -like  prominences. 
Squamose,  scaly,  covered  with  scales. 
Echinate,  beset  with  pointed  prominences. 
Papillate,  beset  with  nipple  or  mamma-like  prominences. 
Rugose,  short,  irregular  folds,  due  to  shrinkage  of  surface  growth. 
Corrugated,  in  long  folds,  due  to  shrinkage. 
Contoured,  an  irregular  but  smoothly  undulating  surface,  like  the  surface  of  a 

relief  map. 
Rimose,  abounding  in  chinks,  clefts,  or  cracks. 


180 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


(C)  Internal  Structure  of  Colony  (Microscopic) : 

1.  Refraction  weak,  outline  and  surface  of  relief  not  strongly  denned. 

2.  Refraction  strong,   outline  and  surface  of  relief  strongly  denned;  dense,   not 
filamentous  colonies. 


FIG.  53.— SHAPES  OF  COLONIES. — (After  Chester.'] 
A.  Cochleate.     B.   Ameboid.      C.  Rhizoid.     F.  Curled. 

General : 

Amorphous,  without  definite  structure  as  below  specified. 
Hyaline,  clear  and  colorless. 

Homogeneous,  structure  uniform  throughout  all  parts  of  the  colony. 
Homochromous,  colony  uniform  throughout  in  color. 


FIG.  54. — SHAPES  OF  COLONIES. — (After  Chester.} 

D.   Mycelioid.     E.   Filamentous. 

• 

Granulations  or  Blotchings: 
Finely  granular. 
Coarsely  granular. 

Grumose,  coarser  than  the  preceding ;  a  clotted  appearance ;  particles  in  clustered 
grains  (Fig.  55,  D). 


182 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


Moruloid,  having  the  character  of  a  morula,  segmented,  by  which  the  colony 

is  divided  in  more  or  less  regular  segments  (Fig.  55,  E). 
Clouded,  having  a  pale  ground,  with  ill-defined  patches  of  a  deeper  tint  (Fig. 

55,  F). 
Colony  Marking  or  Striping: 

Reticulate,  in  the  form  of  a  network,  like  the  veins  of  a  leaf  (Fig.  55,  G). 
Areolate,  divided  into  rather  irregular,  or  angular,  spaces  by  more  or  less  definite 

boundaries. 

Gyrose,  marked  by  wavy  lines,  indefinitely  placed  (Fig.  55,  I). 
Marmorated,  showing  faint,  irregular  stripes,  or  traversed  by  vein-like  markings, 

as  in  marble  (Fig.  55,  H). 

Rivulose,  marked  by  lines,  like  the  rivers  of  a  map. 
Rimose,  showing  chinks,  cracks,  or  clefts. 


FIG.  55.— SURFACE  CHARACTERS  OF  COLONIES.— (After  Chester.] 

A.   Conglomerate.     B.  Toruloid.      C.  Alveolate.     D.  Grumose.     E.  Moruloid.     F.  Clouded. 
G.   Reticulate.     H.   Marmorated.     /.   Gyrose. 


Filamentous  Colonies'. 

Filamentous,  as  already  defined  (Fig.  54,  E). 

Floccose,  composed  of  filaments,  densely  placed. 

Curled,  filaments  in  parallel  strands,  like  locks  or  ringlets,  as  in  agar  colonies 

of  B.  anthracis  (Fig.  53,  F). 
(D)  Edges  of  Colonies: 

Entire,  without  toothing  or  division  (Fig.  56,  A) 

Undulate,  wavy  (Fig.  56,  B). 

Repand,  like  the  border  of  an  open  umbrella  (Fig.  56,  C). 

Erose,  as  if  gnawed,  irregularly  toothed  (Fig.  56,  I). 

Lobate  (Fig.  56,  D). 

Lobulate,  minutely  lobate  (Fig.  56,  D). 

Auriculate,  with  ear-like  lobes  (Fig.  56,  E). 

Lacerate,  irregularly  cleft,  as  if  torn  (Fig.  56,  F). 


184 


LABORATORY  EXERCISES  IN  BACTERIOLOGY 


Fimbriate,  fringed  (Fig.  56,  G). 

Ciliate,  hair-like  extensions,  radiately  placed  (Fig.  56,  H). 
Tufted. 

Filamentous,  as  already  defined. 
Curled,  as  already  defined. 
(E)  Optical  Characters  (after  Shuttleworth) 
Transparent,  transmitting  light. 
Vitreous,  transparent  and  colorless. 

Oleaginous,  transparent  and  yellow;  olive  to  linseed-oil  colored. 
Resinous,  transparent  and  brown;  varnish  or  resin-colored. 
Translucent,  faintly  transparent. 
Porcelaneous,  translucent  and  white. 


F  G  h  I 

FIG.  56. — CHARACTERS  OF  EDGES  OF  COLONIES. — (After  Chester.} 

A.   Entire.     B,   Undulate.      C.   Repand.     D.   Lobate-lobulate.     E.  Auriculate.     F.  Lacerate. 
G.  Fimbriate.     H.   Ciliate.     /.   Erose. 


Opalescent,  translucent,  grayish-white  by  reflected  light,  smoky  brown  by  trans- 
mitted light. 

Nacreous,  translucent,  grayish-white,  with  pearly  luster. 
Sebaceous,  translucent,  yellowish  or  grayish-white. 
Butyrous,  translucent  and  yellow. 
Ceraceous,  translucent,  and  wax-colored. 
Opaque. 

Cretaceous,  opaque  and  white,  chalky. 
Dull,  without  luster. 
Glistening,  shining. 
Fluorescent. 
Iridescent. 


!3 


LESSON  VII. 

STUDY   OF  INDIVIDUAL   BACTERIA,  THEIR  PHYSICAL 
AND   CHEMICAL   CHARACTERISTICS. 

For  the  examination  of  individual  bacteria  one  should  make  use  of  a  good  micro- 
scope fitted  not  only  with  the  ordinary  low-magnifying  powers  (50  to  500  diameters), 
but  also  with  a  clear,  flat  objective  giving  with  the  usual  oculars  and  elongation  of  the 
tube  an  amplification  of  seven  hundred  and  fifty  to  one  thousand  diameters,  and  with 
a  substage  bearing  an  Abbe  light -condensing  apparatus  (to  which  an  iris  diaphragm 
should  be  attached  for  a  convenient  regulation  of  light).  The  student  of  bacteriologic 
technique  may  be  supposed  to  have  become  familiar,  from  his  previous  histologic 
or  biologic  work,  with  the  use  of  the  microscope  ordinarily,  and  for  this  reason 
no  discussion  of  the  principles  involved  in  its  construction  and  manipulation  need 
be  entered  into  here.  It  may  be  opportunely  suggested,  however,  that  in  the  examina- 
tion of  fresh,  unstained  bacteriologic  specimens  the  diaphragm  of  the  light  apparatus 
should  be  contracted  as  much  as  possible  consistent  with  necessary  illumination 
in  order  to  bring  into  sharp  definition  the  outlines  of  the  microorganisms,  but  that 
for  stained  preparations  the  field  should  be  flooded  with  light.  The  higher  objectives 
(one-tenth  or  one-twelfth  inch  focal  distance)  in  common  use  are  oil-immersion  lenses, 
a  drop  of  an  oil  of  refractive  index  nearly  that  of  the  glass  of  the  lens  and  of  the  cover 
and  slide  (thickened  oil  of  cedar  is  usually  used)  being  placed  on  the  preparation,  into 
which  the  tip  of  the  lens  dips  in  focussing,  in  order  to  prevent  loss  of  light.  The 
rays  would  otherwise  be  diverged  on  passing  from  the  glass  of  the  preparation  into  the 
air  between  the  cover  and  the  lens  and  would  be  lost,  and  the  accommodation  for 
light  in  these  short  focal  distance  lenses  is  so  small  that  all  the  rays  must  be  conserved 
for  efficiency.  The  amplification  afforded,  of  course,  does  not  depend  only  upon  the 
lens,  but  as  well  upon  the  eyepiece  of  the  instrument ;  but  the  most  definite  and  clear 
images  are  to  be  obtained  with  the  objectives  of  short  focal  distance  and  oculars  of 
low  power  and  high  light  capacity.  With  such  an  instrument  one  is  enabled  to  make 
all  of  the  observations  necessary  for  ordinary  bacteriologic  investigations;  yet  there 
are  organisms,  as  those  met  by  Roux  and  Nocard  in  the  infectious  pleuropneumonia 
of  cattle,  which  require  for  their  appreciation  a  much  higher  magnification  than  is 
possible  from  the  lenses  suggested  (these  are  barely  recognizable  with  an  amplification 
of  two  thousand  diameters) ;  and  the  difficulty  of  recognition  of  the  agencies  in  yellow 
fever  and  other  infectious  diseases  of  man  makes  it  quite  reasonable  to  suspect  that 
the  failure  to  detect  them  heretofore  has  largely  depended  on  the  minute  size  of  the 
organisms  themselves. 

When  using  a  low-power  lens  (one  inch  or  one-half  inch  focal  distance)  it  is  not 
essential  that  the  material  placed  upon  the  slide  be  covered  with  the  usual  cover-slip, 
all  that  is  essential  being  that  it  be  arranged  jn  an  even  moist  film  through  which  the 
light  may  penetrate  with  uniformity.  So,  too,  in  examination  of  films  which  have  been 

186 


188  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

dried  and  fixed  on  the  slide,  a  drop  of  immersion  oil  may  be  applied  directly  to  the 
film  without  first  adjusting  a  cover-glass  if  the  oil-immersion  lens  be  employed.  In 
all  other  cases  a  thin,  clear  cover  is  essential  for  every  microscopic  preparation. 

After  using  an  oil-immersion  lens  the  oil  should  be  removed  from  the  surface 
of  the  lens  as  well  as  from  the  preparation  by  a  bit  of  soft  absorbent  paper  furnished 
by  dealers  under  the  name  of  "lens  paper"  (dental  absorbent  paper).  If  the  oil  has 
become  dried  upon  a  preparation  a  bit  of  the  paper  moistened  in  xylol  will  be  found 
efficient  for  its  removal;  but  xylol  or  any  solvent  should  be  used  with  extreme  caution 
in  cleaning  an  objective  lest  it  remove  the  balsam  settings  of  the  lenses  and  thus  impair 
the  whole  apparatus. 

Slides  and  Covers. — The  slides  made  of  soft  white  glass  now  usually  sold  by 
dealers  are  on  the  whole  the  best ;  but  in  warm,  moist  climates  they  are  very  liable  to 
corrosion,  especially  if  kept  packed  closely,  and  in  such  localities  the  green  glass  regarded 
as  inferior  is  for  common  use  preferable.  Especial  attention  should  be  had  that  the 
slides  are  perfectly  flat  and  free  from  flaw. 

Covers  should  be  of  white  glass,  very  thin  (No.  1  of  the  manufacturer),  and  their 
surfaces  true.  Squares,  five-eighths  or  three-fourths  of  an  inch  in  size,  will  be  found 
most  convenient.  Like  the  slides,  they  should  not  be  kept  in  close  packets  lest  they 
become  opaque  from  corrosion. 

New  slides  and  covers  should  be  unpacked,  soaked  for  several  hours  in  equal 
parts  of  water  and  one  of  the  strong  mineral  acids,  well  rinsed  in  several  changes  of 
clean  water,  and  kept  in  closed  dishes  in  alcohol  to  which  a  little  ammonia  has  been 
added.  Old  slides  and  covers  may  be  easily  cleaned  after  being  allowed  to  soak  for 
some  hours  or  several  days  in  equal  parts  of  strong  ammonia  and  water ;  after  cleansing 
they  should  be  treated  as  new  slides,  being  soaked  for  several  hours  in  a  dilute  acid 
well  rinsed  in  clean  water,  and  placed  for  keeping  in  dishes  of  ammoniated  alcohol. 
It  is  best  to  keep  old  slides  and  covers  in  dishes  separate  from  those  in  which  are  kept 
the  new  supply. 


MICROSCOPIC   EXAMINATION  OF    CULTURES. 

Colonies  of  bacteria  growing  in  films  of  the  nutrient  on  plates  or  in  dishes  or  rolled 
tubes  are  to  be  examined  with  the  low  powers  of  the  microscope  (twenty  to  sixty 
diameters)  in  order  to  study  their  internal  structure  as  to  refraction,  granulation, 
markings,  etc.  (see  Lesson  VI).  For  this  purpose  the  plate,  dish,  or  tube  is  placed 
directly  on  the  stage  of  the  instrument,  and  observation  made  with  field  darkened 
as  much  as  permissible,  by  contraction  of  the  diaphragm ;  and  subsequently  reflected 
light  should  also  be  used,  the  light  from  the  substage  mirror  being  entirely  cut  off  and 
the  rays  from  some  white  cloud  in  the  sky  concentrated  on  the  surface  of  the  prepara- 
tion by  mirror  or  bull's-eye  condenser.  For  such  examination  it  is  best  to  have  the 
culture  entirely  exposed,  and  for  this  reason  the  cover  of  the  Petri  dish  is  removed; 
but  where  such  exposure  is  objectionable  much  may  be  distinguished  through  the  glass 
of  the  cover,  or  better  through  the  bottom  of  the  dish  which  has  been  turned  over. 

A  favorite  device  used  in  watching  the  development  of  a  colony  of  bacteria  and 
noting  its  minute  features  is  the  "hanging-drop"  preparation,  also  employed  in  high- 
power  studies  of  the  motility,  grouping,  spore-formation  and  germination,  agglutina- 
tion, etc.,  of  bacteria.  A  hanging  drop  (Fig.  57)  requires  a  special  slide,  in  the  upper 
surface  of  which  a  small  concavity  has  been  ground  and  polished.  A  clean  cover-glass 
having  been  procured,  a  drop  of  physiologic  salt  solution,  bouillon,  or  of  liquefied  agar  or 


190  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

gelatine  is  placed  on  the  under  surface,  and  with  the  point  of  the  platinum  needle 
some  of  the  organisms  to  be  studied  transferred  to  the  drop.  A  thin  ring  of  vaseline 
is  drawn  about  the  margin  of  the  concavity  in  the  slide  with  a  hair  pencil  by  which, 
when  the  cover  is  applied,  with  the  drop  suspended  in  the  slide  space  from  its  under 
surface,  it  may  be  retained  in  position,  drying  prevented,  and  the  danger  of  contamina- 
tion from  the  outside  removed.  When  it  is  intended  to  observe  the  development 
of  the  colonies  of  bacteria  growing  in  the  inclosed  drop,  the  slide  and  cover  should, 
of  course,  have  been  previously  sterilized  (in  the  flame)  and  the  medium  used  should 
be  gelatine  or  agar,  the  whole  preparation  then  being  submitted  to  the  usual  condi- 
tions of  temperature  and  atmosphere  (if  grown  in  anaerobic  jar,  the  vaseline  ring 
should  not  have  been  complete,  to  allow  the  replacement  of  air  in  slide  chamber  with 
hydrogen  or  nitrogen)  as  for  any  other  culture  preparation.  Where  prolonged  ob- 
servation of  a  selected  field  is  desirable  during  a  period  of  incubation,  special  incubators 
permitting  the  introduction  of  the  microscope  into  the  interior,  and  allowing  through 


FIG.  57. — DIAGRAM  OF  THE  HANGING  DROP. 

glass  sides  the  entrance  of  light,  are  sold ;  but  if  one  makes  the  examination  in  a  warm 
room  and  uses  the  mechanical  stage  for  the  location  of  the  desired  field,  this  rather 
cumbersome  and  costly  device  is  not  required.  In  studies  of  the  individual  germs 
requiring  less  prolonged  observation  the  loopful  of  salt  solution  or  bouillon  inoculated 
from  the  desired  growth  (or  a  loopful  of  an  original  liquid  culture)  is  usually  used 
instead  of  the  solid  media,  and  there  is  no  need  beyond  reasonable  cleanliness  for 
sterilization.  After  use  of  such  a  slide  and  cover  they  should  be  sterilized  and  cleansed 
by  one  of  the  methods  prescribed  for  other  containers. 

Care  must  always  be  observed,  in  the  use  of  the  high  power  with  such  a  prepara- 
tion, from  the  danger  of  breaking  the  unsupported  cover-slip  by  inadvertent  pressure 
of  the  lens  upon  it  in  focussing.  The  edge  of  the  hanging  drop  should  be  adjusted 
with  the  low  power,  and  thereafter  the  higher  power  substituted  and  the  edge  cautiously 
focussed,  after  which  it  is  usually  easy,  with  the  fine  adjustment  and  movement  of  the 
slide,  to  obtain  the  desired  field  in  proper  focus. 

EXAMINATION  OF  INDIVIDUAL  BACTERIA. 

A  loopful  of  physiologic  salt  solution  or  bouillon,  or  even  clean  water,  may  be 
placed  on  a  clean,  plain  slide,  a  few  bacteria  from  some  culture  transferred  and  diffused 
by  drawing  the  needle  once  or  twice  through  the  drop  (or  a  loopful  of  a  culture  in  any 
of  the  liquid  media  may  be  used  instead).  A  cover  is  then  adjusted  and  the  preparation 
at  once  examined  (with  moderately  dark  field)  under  the  higher  power  of  the  micro- 
scope. In  such  case  the  arrangement  of  the  individual  organisms  is,  of  course,  more 
or  less  lost,  the  hanging  drop  being  preferable  when  the  latter  feature  is  sought  to  be 
studied.  But  examination  of  fresh,  unstained  material  in  this  manner  or  by  the 
hanging  drop  is  essential  for  determination  of  the  motility  of  the  organisms.  Neither 
is  comparable  to  stained  preparations  for  the  most  exact  study  of  the  morphology 
of  the  individual  germs.  In  the  latter  case  the  material  must  before  being  stained  be 
obtained  in  a  thin  film  ("smear")  upon  the  slide  or  cover. 


192  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

Smears  for  Stained  Preparations. — The  film  may  be  spread  upon  either  a 
clean  slide  or  cover-slip,  the  former  being  much  preferable  in  that  it  is  more  conveniently 
handled  and  is  in  less  danger  of  being  broken.  The  writer  usually  places  the  film  a 
little  to  one  side  of  the  center  of  the  slide  (not  so  far  as  to  interfere  with  centering  on  the 
mechanical  stage),  and  is  thus  relieved  of  the  necessity  of  using  a  forceps  for  handling 
the  preparation.  When  made  on  a  cover  it  is  essential  to  use  one  of  the  different  forms 
of  forceps  (cover-glass  holder),  as  that  of  Stewart  (Fig.  58),  in  order  to  pursue  the  work 
without  danger  to  the  preparation  or  of  getting  the  germs  upon  the  fingers. 

The  surface  of  the  slide  or  cover  must  be  entirely  clean  and  quite  free  from  traces 
of  grease  which  are  apt  to  come  on  the  glass  from  handling.  When  taken  from  the 
ammoniated  alcohol  in  the  tray  the  slide  or  cover  should  be  carefully  dried  with  an 
old  linen  rag  which  is  quite  free  from  grease  from  inferior  soap  or  other  source,  or  with 
a  bit  of  clean,  unsized,  unfilled  paper.  A  drop  of  water  on  the  surface  should  spread 
out  flat  and  evenly  and  not  separate  into  small  droplets  or  persist  in  globular  form 
when  it  is  attempted  to  draw  it  over  the  surface  with  the  needle.  Should  the  drop 
refuse  to  spread  readily  it  may  be  concluded  that  the  glass  is  not  entirely  clean;  and 
in  such  case  it  is  best  to  wash  it  with  good  soap  and  water,  and  then  rinse  it  first  in 
water  and  then  in  alcohol,  and  again  dry  it  with  paper  or  a  clean  rag. 

(a)  In  examination  of  cultures  it  is  preferable  to  obtain  the  germs  from  a  growth 
on  a  solid  medium,  the  bouillon  in  a  film  rendering  uniform  staining  difficult.  A  small 


FIG.  58. — STEWART  FORCEPS  FOR  COVER-GLASSES. 

drop  of  clean  water  is  placed  in  proper  position  on  a  slide  or  cover.  The  platinum 
needle,  having  been  flamed,  is  used  to  obtain  a  trace  of  the  culture  to  be  examined, 
which  is  then  diffused  in  the  drop  until  a  very  faint  turbidity  is  noticed  (best  seen  in 
a  side  light) .  With  the  needle  the  water  with  its  contained  bacteria  is  now  spread  over 
a  convenient  area,  the  needle  at  once  flamed  to  destroy  the  infection  remaining  on  it 
and  the  film  allowed  to  dry  in  the  air.  (If  a  cover  was  used  the  film  might  have  been 
spread  by  application  of  a  second,  clean  cover,  the  two  after  close  adjustment  being 
slipped  apart  so  as  to  evenly  distribute  the  fluid  over  the  adjacent  surfaces  of  the  two 
covers.  In  this  case  use  forceps  in  handling.)  The  drying  is  completed  and  the  film 
fixed  to  the  surface  of  the  slide  by  passing  the  latter  by  a  slow,  even  movement  through 
the  flame  of  a  Bunsen  burner  or  spirit  lamp  several  times,  the  film  on  the  upper  side 
and  away  from  the  flame,  to  prevent  scorching.  Usually,  three  passages  through  the 
flame  will  suffice ;  the  glass  should  be  distinctly  hot,  but  not  sufficiently  to  be  actually 
painful  when  touched  to  the  back  of  the  hand.  Thus  prepared  the  film  should  be 
visible  as  a  delicate  opacity,  with  dead,  non-glistening  surface.  It  is  now  ready  for 
staining  or  may  be  kept  indefinitely  (placed  with  film  upward  on  a  sheet  of  paper 
bearing  proper  label  and  covered  over  with  suitable  cover  to  keep  the  atmospheric 
dust  from  settling  on  it — or  in  a  small  paper  box). 

(6)    Smears   of   Organic   Fluids   Containing   Bacteria. — Fluids  containing  organic 
matter  in  which  bacteria  are  diffused  are  made  into  similar  smear  preparations  by 


194  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

being  added  in  small  proportions  to  a  drop  of  water  on  a  slide  or  cover.  Thus,  one 
should  draw  the  loop  filled  with  a  bouillon  or  milk  culture  through  a  large  drop  of 
distilled  water  in  a  watch-crystal  first;  then,  having  flamed  the  loop,  a  small  amount 
of  this  drop  is  transferred  to  the  drop  of  water  on  the  slide  and  again  slightly  diffused. 
The  organic  matter  interferes  more  or  less  with  the  even  distribution  of  the  film,  and 
when  dried  prevents  a  uniform  penetration  of  the  stain ;  for  which  reasons  the  dilution 
suggested  is  to  be  practised.  The  remaining  steps  in  spreading  and  fixing  the  film 
are  the  same  as  above  when  the  material  was  obtained  directly  from  a  mass  of  bacteria 
on  a  solid  medium. 

The  exudates  and  fluid  secretions  and  excretions  obtained  from  diseased  individuals, 
as  sputum,  pus,  or  urine,  are  usually  spread  in  films  without  dilution.  When  mucus 
is  present  in  such  substance,  preventing  easy  spreading,  it  is  well  to  warm  the  slide 
slightly  and  insure  an  even  distribution  in  a  thin  film  by  pressing  a  second  slide  upon 
the  drop  and  drawing  it  across  the  surface  of  the  first  slide.  This  done,  the  film  is 
dried  in  the  air  and  fixed  by  heat  as  above.  Where,  as  in  case  of  urine,  there  is  much 
saline  matter  present,  it  is  best  to  remove  it  before  naming  the  slide  for  fixation  of  the 
film  by  placing  the  slide  for  a  few  minutes  in  a  vessel  with  excess  of  water,  by  which 
these  substances  will  be  taken  up.  It  is  again  dried  in  the  air  and  the  film  fixed  in  the 
flame.  It  is  then  ready  for  immediate  application  of  the  stain,  or  may  be  kept  as 
above  indefinitely. 

(c)  Smears  from  Tissues. — After  inoculations  have  been  made  from  foci  of  disease 
in  tissues  obtained  by  surgical  operation  or  autopsy,  a  section  should  be  made  with  a 
clean  knife  and  the  cut  surface  of  the  tissue  drawn  lightly  and  evenly  over  the  slide. 
The  resulting  film  is  dried  in  the  air  and  fixed  either  in  the  flame  or  by  being  immersed 
for  five  or  ten  minutes  in  a  mixture  of  equal  parts  of  absolute  alcohol  and  ether.     It 
may  then  be  at  once  stained  or  kept  indefinitely  in  the  fluid  or  dry.     The  same  pro- 
cedures are  applicable  when  the  material  has  been  obtained  on  a  swab  of  cotton,  as 
diphtheritic  exudate,  the  swab  being  wiped  lightly   over  the  surface  of  the  slide  or 
cover. 

(d)  Impression  Films. — These  are  prepared  by  pressing  gently  and  evenly  a  clean, 
dry  cover  or  slide  to  the  surface  of  a  colony  growing  on  the  surface  of  some  solid  medium. 
After  application,  it  is  raised,  without  sliding,  directly  from  the  colony,  a  print  or  im- 
pression being  retained  from  the  adhesion  to  the  glass  of  the  superficial  individuals 
in  the  growth.     As  before,  the  film  is  dried  in  the  air  and  fixed  in  the  flame.     This  pro- 
cedure is  intended  for  the  preservation  of  the  relations  of  the  individual  bacteria  to 
each  other  in  the  colony,  some  organisms  in  their  growth  producing  extremely  char- 
acteristic arrangements,  often  in  marvelous  patterns  (Bact.  mycoides,  vel  figurans). 


STAINING  REAGENTS  AND  MIXTURES. 

The  difficulty  of  exact  appreciation  of  the  isolated  bacterium  is  much  diminished 
by  proper  staining,  for  which  purpose  the  basic  anilines  are  most  efficient.  The  ordinary 
nuclear  stains  used  in  histologic  work  have  little  penetrative  power  for  bacteria,  but 
if  intensely  applied  are  sometimes  capable  of  tinging  the  bacterial  substance.  How- 
ever, their  use  is  attended  with  such  difficulties  and  the  results  of  such  moderate  value 
that  they  are  practically  never  used,  the  anilines  being  employed  almost  exclusively. 
Basic  fuchsin,  gentian  violet,  methylene-blue,  thionine,  and  a  few  others  are  the  most 
commonly  used.  Aqueous  solutions  are  the  most  efficient,  the  dried  bacteria  in  the 
film  preparations  permitting  their  diffusion  in  the  bacterial  substance  to  a  far  greater 


196  LABORATORY  EXERCISES  IN  BACTERIOLOGY 

degree  than  alcoholic  solutions,  which  for  the  most  part  are  employed  only  as  counter- 
stains  or  as  stock  solutions  from  which  the  watery  solutions  are  prepared  as  required. 
There  is  little  difference  manifested  by  different  types  of  vegetating  bacteria  in  the 
assumption  of  the  staining  reagents,  but  some  types  require,  in  addition  to  the  simple 
solution  of  the  dye,  the  previous  or  associated  influence  of  substances  like  the  alkalies, 
aniline  oil,  acetic  acid,  or  carbolic  acid  for  the  proper  penetration  of  the  coloring  matter. 
Such  a  substance  is  known  as  a  mordant.  Its  precise  mode  of  action  is  not  clearly 
understood  and  probably  is  not  in  all  instances  identical.  It  may  involve  the  removal 
of  some  oleaginous  material  from  the  bacterial  capsule  or  the  production  of  chemical 
alterations  in  the  composition  of  the  latter,  by  which  it  becomes  more  pervious  to  the 
staining  fluid  and  thus  allows  the  latter  to  enter  the  protoplasmic  body  of  the  bac- 
terium. The  organisms  of  tuberculosis  and  leprosy  and  the  mycobacterium  of  smegma 
are  prominent  examples  which  require  the  addition  of  such  a  mordant  for  effective 
staining;  and  the  spores  of  all  bacteria  show  extreme  refractiveness  to  the  penetration 
of  stains,  even  in  the  presence  of  the  common  mordants. 

As  a  group  bacteria  are  not  so  readily  colored  as  are  animal  structures,  and  in 
fact  there  is  some  difference  between  different  varieties;  and,  as  is  the  usual  rule  that 
such  substances  as  are  with  difficulty  colored  may  be  not  readily  decolorized,  they  are 
apt  to  retain  their  coloration  in  the  presence  of  decolorizing  agents  longer  than  asso- 
ciated animal  matter,  which  has  been  quickly  and  easily  stained.  So,  too,  some 
difference,  available  in  a  few  instances  for  diagnostic  purposes,  between  different  types 
subjected  to  a  definite  decolorizer  may  be  distinguished.  Of  these  decolorizing  agents 
water  is  the  type,  but  the  least  active ;  its  power  of  removal  of  stains  is  increased  when 
it  is  used  hot.  Alcohol  is  more  energetic,  a  few  minutes  of  exposure  of  stained  films 
being  sufficient  to  discharge  the  tint  from  animal  tissues  as  well  as  from  many  bacteria. 
Among  the  more  powerful  decolorizing  agents  in  common  use  are  weak  solutions  of 
acetic  acid  or  of  the  mineral  acids. 

The  process  of  staining  the  films  as  above  prepared  is  usually  carried  out  by  placing 
a  few  drops  -of  the  coloring  solution  on  the  film  fixed  upon  the  surface  of  the  slide  or 
cover-glass  (film  side  uppermost),  contact  being  continued  for  from  a  half  minute  to 
several  hours  in  case  of  different  organisms,  varying  with  the  type  of  organism,  the 
staining  fluid  used,  and  the  application  of  heat  (the  process  being  much  shortened  when 
the  stain  is  applied  hot).  If  one  prefers,  a  cover  preparation  may  be  floated  upon  the 
surface  of  the  staining  fluid,  the  film  side  downward,  in  contact  with  the  stain.  Every- 
thing capable  of  being  colored  by  the  stain  having  been  sufficiently  tinged,  the  prepara- 
tion is  rapidly  washed  through  water  or  one  of  the  other  decolorizing  fluids  so  as  to 
discharge  the  color  from  everything  in  the  film  but  the  bacteria.  Where  several  types 
of  organisms  of  different  resistance  to  decolorizations  exist  in  the  preparation,  the 
differentiation  is  accomplished  by  permitting  sufficient  action  of  the  decolorizer  to 
remove  the  stain  from  the  less  resistant,  but  not  sufficient  to  accomplish  the  decoloriza- 
tion  of  the  more  resistant.  As  a  third  step  of  the  process,  one  may,  for  greater  ease  of 
detection  of  the  stained  organisms  in  the  preparation,  quickly  apply  a  contrast  stain  to 
those  elements  from  which  the  decolorizing  fluid  has  discharged  the  first  stain.  Eosin, 
vesuvin,  methylene-blue,  safranin,  and  Bismarck  brown  in  alcoholic  solution  are  usually 
chosen  for  this  purpose. 

These  aniline  stains  are  not  so  permanent  as  hematoxylin  or  carmine  stains; 
preparations  are  best  preserved  if  kept  in  closed  boxes  away  from  the  light. 


198  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

STAINING    SOLUTIONS. 

1 .  There  should  be  kept  in  the  laboratory  supplies  of  the  following  stock  solutions : 
saturated  alcoholic  solutions  of   (a)  basic  fuchsin,  (6)  gentian  violet,  (c)  methylene-blue, 
(d)  thionine,  (e)  eosin,  (/)  vesumn.      These  are  prepared  from  strong  alcohol  and  an 
excess  of  the  stain;  as  used,  more  alcohol  may  be  added,  always  with  the  caution  that 
some  excess  of  undissolved  stain  remains.     Each  student  has  in  his  locker  his  individual 
tray  of  tubes  (Fig.  59),  in  which  watery  solutions  of  the  above  are  kept.     One  cubic 
centimeter  of  the  saturated  alcoholic  solution  of  aniline  dye  to  ten  or  twelve  cubic 
centimeters  of  distilled  water  are  used  in  the  preparation  of  the  latter  aqueous  solutions. 
Each  tube  is  provided  with  tubules  as  in  a  chemical  wash-flask,  for  discharge  of  the 
stain  as  needed,  a  wad  of  absorbent  cotton  in  the  lower  end  of  the  exit  tubule  serving 
as  a  filter.     Owing  to  the  fact  that  these  watery  solutions  quickly  deteriorate,  they 
should  not  be  used  if  old,  and  should  never  be  made  up  in  large  quantities. 

2.  The  following  special  stains  and  other  fluids  frequently  used  in  staining  are  also 
to  be  kept  in  stock: 

(a)  Ziehl's  Carbol-fuchsin: 

Fuchsin, I 

Absolute  alcohol, 10 

Five  per  cent,  aqueous  sol.  carbolic  apid, 90 


FIG.  59. — TRAY  OF  STAINING  TUBES. 

Used  in  staining  mycobacterium  of  tuberculosis,  etc.  This  solution  keeps  well  for 
several  months.  The  carbolic  acid  in  its  composition  is  added  as  a  mordant.  In 
preparation  throw  the  fuchsin  on  a  filter  paper  and  after  mixing  the  alcohol  and  the 
acid  solution  percolate  until  the  stain  is  all  dissolved.  Filter  before  use.  Contact  with 
films  four  or  five  minutes  if  warm,  longer  if  cold. 

(6)  Gabbet's  Solution: 

Methylene-blue, 2 

Twenty-five  per  cent.  aq.  sol.  of  sulphuric  acid, 100 

Used  as  decolorizing  agent  and  as  counterstain  after  carbol-fuchsin.  Acid  solution 
should  be  cooled  before  being  added  to  the  stain.  In  preparation  throw  stain  on  a 
filter  paper  and  percolate  the  acid  solution  until  the  stain  is  all  dissolved.  This  solution 
is  not  liable  to  decomposition.  Filter  before  use.  Contact  with  film,  cold,  for  from 
half  a  minute  to  two  minutes. 

(c)  Loe filer's  Blue  Solution: 

Sat.  ale.  sol.  methylene-blue, 3° 

I  :  10,000  aq.  sol.  potassium  hydroxide, IO° 


200  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

Used  for  general  staining,  but  especially  for  gonococcus  and  mycobacterium  of 
diphtheria.  This  solution  is  permanent ;  the  alkali  serves  as  mordant.  Filter  before 
use.  Contact  with  film  from  half  to  several  minutes,  warm  or  cold  as  required  in  each 
case. 

(d)  Ehr lick's  Aniline-water  Solution: 

Aniline  oil, 5 

Distilled  water, 100 

Shake  well  the  above  ingredients  in  mixture  at  intervals  for  five  or  ten  minutes  to 
favor  solution  of  the  oil.  Filter  through  a  moist  filter  paper.  To  each  ten  cubic 
centimeters  of  this  "aniline  water"  add  freshly  one  cubic  centimeter  of  saturated 
alcoholic  solution  of  gentian  violet  or  fuchsin  for  use.  The  aniline  water  does  not  keep 
well  and  should  be  renewed  in  the  stock  bottle  every  week  or  ten  days.  The  mixed 
stain  also  keeps  poorly  and  should  be  made  fresh  every  few  days,  and  filtered  before 
use.  These  solutions  are  used  as  general  stains  or  for  special  differential  purposes  in 
Gram's  method;  they  are  useful  in  demonstration  of  bacteria  in  sections  of  tissues. 

In  conjunction  with  these  solutions  in  Gram's  method  the  following  decolorizing 
solution  is  employed: 

(e)  Gram's  Differential  Solution: 

Iodine, I 

Potassium  Iodide, 2 

Water, 300 

Pulverize  potassium  iodide  in  a  mortar ;  dissolve  in  water  and  add  iodine,  frequently 
shaking  to  accomplish  solution.     Filter.     The  solution  keeps  well. 
(/)  Carbol-thionine : 

Thionine, I 

2.5  per  cent.  aq.  sol.  carbolic  acid, 100 

Used  as  a  general  stain;  valuable  as  a  stain  for  bacteria  in  tissues.  Add  equal 
proportions  of  water  for  staining.  Filter  before  use.  Keeps  well.  Stain  three  to 
five  minutes.  Decolorize  with  alcohol. 

(g)  Unna's  Polychrome  Blue: 

Methylene-blue, I 

Potassium  carbonate, I 

Water, 100 

This  is  used  in  staining  bacteria  in  tissues.     Should  be  ripened  for  months  before 
use.     Permanent.     Contact  five  or  more  minutes. 
(h)  Acid  Alcohol: 

Hydrochloric  acid, 3 

Seventy  per  cent,  alcohol, 97 

This  is  stronger  than  the  acid  alcohol  used  in  histologic  work.  Used  as  a  decoloriz- 
ing agent. 

(i)  Nitric  Acid  Solution: 

Nitric  acid, 30 

Water, 7° 

Used  as  a  decolorizing  agent  for  'Mycobacterium  tuberculosis,  etc. 


14 


202  LABORATORY  EXERCISES  IN  BACTERIOLOGY, 

(k)  Acetic  Acid  Solution: 

Acetic  acid  (glacial),     ... 2 

Water, 100 

Sometimes  used  as  a  mordant,  the  film  preparation  being  flooded  with  the  solution 
for  half  a  minute,  then  washed  in  water  or  acid  blown  off  with  fine  blowpipe.  A  good 
decolorizing  agent  for  ordinary  stains. 

STAINING  METHODS. 

In  the  study  of  any  unknown  organism  it  should  be  a  rule  to  subject  film  prepara- 
tions first  to  the  simple  aqueous  solution  of  gentian  violet. or  fuchsin,  then  to  Loeffler's 
blue,  to  Gram's  method,  and  finally  to  the  Ziehl's  carbol-fuchsin  and  Gabbet's  blue, 
recording  the  result  of  each  method. 

In  staining  a  film  with  the  ordinary  aqueous  solutions  it  is  customary  to 
flood  the  film  with  a  few  drops  of  the  staining  fluid.  Usually  there  is  no  need  of  heating ; 
but  should  the  stain  be  taken  up  but  slowly,  the  slide  or  cover  may  be  held  over  the 
flame  until  vapor  arises  (not  boiled).  The  staining  is  usually  accomplished,  when  the 
solution  is  warmed,  in  one-half  to  one  minute ;  when  cold,  usually  from  three  to  five 
minutes  are  required.  If  preferred,  the  staining  solution  may  be  placed  in  a  dish  or 
watch-crystal,  and  the  cover  on  which  the  film  is  spread  floated  on  the  surface  of  the 
fluid,  film  side  downward.  After  sufficient  exposure  to  the  stain  the  preparation  is 
held  under  the  tap  in  a  gentle  stream  of  water  or  waved  gently  through  a  dish  of  water 
until  color  is  no  longer  discharged.  This  is  usually  sufficient;  but  should  the  film 
retain  a  deep  tint,  it  is  well  to  wash  for  a  moment  in  alcohol,  and  again  rinse  in  water. 
The  film  is  then  dried  carefully,  being  held  at  some  height  over  the  flame  until  quite 
dry.  It  is  then  to  be  examined  directly  in  oil,  or  a  cover  is  added  with  Canada  balsam, 
as  usual,  and  the  preparation  then  examined  with  the  microscope. 

Practically  all  bacteria  may  be  stained  by  this  method.  The  mycobacterium  of 
tuberculosis  may  be  stained  only  with  such  difficulty,  however,  that  in  ordinary  prepara- 
tions containing  this  organism  it  is  not  found.  So  with  tissues  containing  the  Myco- 
bacterium leprce;  and  the  organisms  of  glanders,  Asiatic  cholera,  and  influenza  also 
stain  very  poorly,  but  may  be  demonstrated  in  deeply  stained  preparations  made  with 
heat. 

Loeffler's  alkaline  blue  is  of  general  value,  but  many  of  the  organisms  take  up 
the  stain  but  faintly.  It  is  especially  useful  for  staining  the  organisms  of  diphtheria 
and  gonorrhea.  The  stain  is  applied  by  flooding  the  film  with  several  drops.  It 
acts  best  if  somewhat  warmed  (not  hot),  and  successful  preparations  are  made  in 
from  a  few  seconds  to  one  or  two  minutes.  The  film  is  then  washed  well  in  water, 
dried,  and  examined  either  after  application  of  balsam  and  cover,  or  directly  in  oil 
{oil-immersion  lens). 

In  the  use  of  Gram's  method  probably  the  most  important  differential  staining 
process  is  met.  For  prosecution  of  this  method  the  film  is  well  stained,  with  use  of 
moderate  warmth,  with  Ehrlich's  aniline-water  solution  of  gentian  violet  or  fuchsin 
for  three  or  four  minutes.  The  excess  of  the  stain  is  washed  in  water,  and  the  film 
is  flooded  with  Gram's  iodine-iodide  solution  for  one  minute,  the  film  now  becoming 
dark.  It  is  next  washed  in  ninety-five  per  cent,  alcohol  until  the  color  is  almost  dis- 
charged. vShould  the  original  tint  of  the  film  return  persistently,  the  preparation  is 
again  treated  with  Gram's  solution  and  again  washed  in  alcohol.  After  almost  total 


204  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

disappearance  of  the  color  the  film  is  dried  and  may  be  examined  in  oil  (oil-immersion 
lens)  or  covered  in  the  usual  manner  before  examination.  Counterstaining  with 
vesuvin  or  eosin  may  be  practised  if  desired. 

The  following  list  includes  the  most  important  forms  which  are  decolorized  by 
this  method,  while  the  important  forms  which  retain  the  stain  may  be  inferred  from 
their  absence  from  the  group  mentioned:  Gonococcus,  Micrococcus  of  Malta  fever, 
Micrococcus  catarrhalis,  Micrococcus  boms,  Micrococcus  magnus,  Micrococcus  Vincenzii, 
Streptococcus  Kirchneri,  Streptococcus  canis,  Bacterium  influenza,  Bacterium  conjunc- 
tivitidis,  Bacterium  cancrosi,  Bacterium  agyptiacum,  Bacterium  chinense,  Bacterium 
Wrightii,  Bacterium  pneumonia  (Friedlander),  Bacterium  cholera,  Bacterium  san- 
guinarium,  Bacterium  avium,  Bacterium  saliva,  Bacterium  ambiguum,  Bacterium 
radiatum,  Bacterium  ovatum,  Bacterium  Lepierrei,  Bacillus  Marsiliensis,  Bacillus  coli, 
Bacillus  Wardii,  Bacillus  anindolicus,  Bacillus  enteritidis,  Bacillus  chologenes,  Bacillus 
toxigenus,  Bacillus  brassica  (?),  Bacillus  icterogenes,  Bacillus  Poelsii,  Bacillus  colum- 
barum,  Bacillus  Breslaviensis,  Bacillus  Salmonii,  Bacillus  levans,  Bacillus  loxiacida, 
Bacillus  morbificans,  Bacillus  Silberschmidii,  Bacillus  Murium,  Bacillus  intestinal  is, 
Bacillus  meningitidis,  Bacillus  typhosus,  Bacillus  pseudotyphosus,  Bacillus  icteroides, 
Bacillus  Billingsi,  Bacillus  paradoxus,  Bacillus  pestis,  Bacillus  solitarius,  Bacillus 
geminus,  Bacillus  aquatilis-sulcatus-quartus,  Bacillus  primus  Fullesi,  Bacillus  trache- 
iphilus,  Bacillus  pinatus,  Bacillus  Ravaneli,  Bacillus  alcaligenes,  Bacillus  Friedber- 
gensis,  Bacillus  solanacearum,  Bacillus  Weichselbaumi,  Bacillus  phasiani,  Bacillus 
Schafferi,  Bacillus  rugosus,  Bacillus  amsepticus,  Bacillus  avium,  Bacillus  meleagris, 
Bacillus  tetraonis,  Bacillus  cygneus,  Bacillus  aerobius,  Bacillus  pneumosepticus,  Bacillus 
monachce,  Bacillus  cuniculi,  Bacillus  venosus,  Bacillus  glischrogenus,  Bacillus  •  albus, 
Bacillus  granulatus,  Bacillus  stolonatus,  Bacillus  invisibilis,  Bacillus  venenosus,  Bacillus 
murinus,  Bacillus  denitrificans,  Bacillus  Stutzeri,  Bacillus  centra punctatus,  Bacillus 
agilis,  Bacillus  Hartlebii,  Bacillus  murisepticus,  Bacillus  Wesenbergii,  Bacillus  larvi- 
cida,  Bacillus  dendriticus,  Bacillus  Kornii,  Bacillus  prodigiosus,  Bacillus  kiliensis, 
Bacillus  licheniformis,  Bacillus  oedematis,  Bacillus  Weigmannii,  Bacillus  saccharo- 
butyricus,  Bacillus  longus,  Pseudomonas  punctata,  Pseudomonas  campestris,  Pseudo- 
monas  pyocyanea  (?),  Pseudomonas  capsulata,  Pseudomonas  fluorescens,  Pseudomonas 
putida,  Microspira  phosphorescens,  Microspira  Schuylkilliensis,  Microspira  comma, 
Microspira  Danubica,  Microspira  Beroliniensis,  Microspira  protea,  Microspira 
aquatilis,  Spirochata  Obermeieri,  Mycobacterium  influenza,  Mycobacterium  Elmassiani, 
Mycobacterium  mallei  (?),  Mycobacterium  hastilis. 

Staining  of  Mycobacterium  Tuberculosis  and  Allied  Forms. — The  myco- 
bacterium  of  tuberculosis  is  especially  resistant  to  staining,  and  having  been  once 
stained  is  equally  resistant  to  decolorization  by  means  of  the  mineral  acids.  In  this 
rests  its  peculiarity  in  differential  staining.  Many  bacteria  respond  nearly  or  quite 
equally  to  this  organism  when  stained  by  Gram's  method,  but  there  are  few  that  are 
able  to  withstand  the  influence  of  the  mineral  acids  employed  for  decolorization  of  the 
stained  preparation.  The  varieties  which  approach  the  tubercular  organism  are  the 
mycobacterium  of  leprosy  and  that  of  smegma,  and  several  forms  of  the  same  group 
isolated  from  butter,  hay,  and  grass.  The  latter  very  closely  simulate  the  organism 
of  tuberculosis ;  but  excluding  these,  the  degree  of  resistance  to  decolorization  by  acids 
and  by  alcohol  serves  to  differentiate  the  others  from  the  cause  of  tuberculosis.  For 
demonstration  of  this  microbe  the  use  of  one  of  the  aniline  dyes  reinforced  with  a 
mordant  is  essential,  and,  with  subsequent  decolorization  of  all  the  elements  in  the 
preparation  save  Mycobacterium  tuberculosis  by  one  of  the  mineral  acids,  affords  a 


206  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

means  of  clinical  diagnosis  from  the  other  bacteria  apt  to  be  met  in  persons  suspected 
of  having  tuberculosis  which  is  practically  reliable.  A  favorite  method  for  determina- 
tion of  these  germs  in  the  sputum  of  consumptives  may  be  detailed  in  illustration : 

1 .  Spread  sputum  on  a  plate  of  glass  over  a  black  surface  and  with  a  forceps  pick 
out  the  small  yellowish  masses,  which  are  likely  to  contain  numbers  of  the  bacteria. 
(Do  not  mistake  particles  of  food!) 

2.  Place  such  material  on  a  glass  slide  and  spread  by  applying  a  second  slide  upon 
the  mass,  drawing  it  along  and  across  the  slide  evenly.     (Warm  if  spreading  is  inter- 
fered with  by  mucus.) 

3.  Dry  film  in  air,  and  fix  by  passing  three  times  through  the  flame,  smeared  side 
away  from  flame.     (Do  not  burn!) 

4.  Flood  film  with  Ziehl's  carbol-fuchsin  (supra,  a),  and  warm  until  vapor  arises 
(do  not  boil!)  over  flame  for  three  or  four  minutes. 

5.  Wash  off  excess  of  stain  under  tap,  and  apply  as  decolorizing  agent  and  counter- 
stain  Gabbet's  solution  (6)  for  one-half  to  one  minute. 

6.  Wash  in  water  until  no  more  color  is  discharged. 

7.  Dry  film  and  examine  directly  in  oil,  or  after  covering  with  cover-slip.     Mrco- 
bacteria  tuberculosis  red ;  all  else  in  field  blue. 

The  examination  of  urine  and  vaginal  discharge  is  similarly  carried  out,  but 
after  discovery  of  acid-resisting  bacteria  one  should  apply  a  saturated  alcoholic  solution 
of  methylene-blue  to  the  film  for  a  minute.  Should  the  bacteria  be  decolorized  and 
stained  blue  by  this  procedure  they  are  not  the  organisms  of  tuberculosis,  but  the 
smegma  mycobacteria,  non-pathogenic  bacteria  found  in  the  cheesy  secretion  about 
the  foreskin  or  vulva. 

Staining  Bacteria  in  Sections  of  Tissues. — Tissues  in  which  bacteria -are  to 
be  stained  should  be  at  once  fixed  in  absolute  alcohol.  After  sections  have  been  made 
in  one  or  other  of  the  usual  manners  (freezing,  paraffine,  or  celloidine  methods — for 
technique  of  which,  consult  any  work  on  histologic  technology)  a  section  should  be 
attached  to  a  slide.  This  may  be  done  by  floating  the  section  on  a  drop  or  two  of  a 
solution  of  gelatine  (best  sheet  gelatine,  0.5;  chloral  hydrate,  1  ;  distilled  water,  100) 
for  several  minutes,  after  which  the  excess  of  the  solution  is  drained  off  and  the  slide 
set  aside  to  dry  (best  until  the  following  day  under  a  bell  jar;  more  quickly  at  37°  C. 
in  incubator).  Thereafter  it  is  plunged  for  five  minutes  in  a  five  per  cent,  solution  of 
potassium  bichromate  to  fix  the  gelatine  glue ,  and  render  it  insoluble  in  the  staining 
fluids  to  be  used.  (Specimens  in  paraffine  should  have  paraffine  removed  prior  to  this 
last  step.)  This  done,  the  staining  is  carried  out  as  if,  instead  of  a  section,  the  ordinary 
film  were  on  the  slide.  It  is  best,  however,  to  use  little  or  no  heat  and  to  allow  the 
stains  a  longer  time  for  action  before  removal  and  application  of  the  decolorizer.  So, 
too,  in  use  of  chemical  decolorizers  the  best  results  usually  follow  the  use  of  the  less 
concentrated  solutions,  and  separate  application  of  the  decolorizer  and  counterstain. 
The  sections  should  be  almost  completely  decolorized  to  gross  inspection.  In  the 
final  dehydration  before  mounting,  absolute  alcohol  should  be  used  drop  by  drop  on 
the  section,  alternating  with  occasional  absorption  of  all  fluid  possible  by  application 
of  a  layer  of  folded  unsized  paper.  Origanum  oil  is  best  suited  as  a  clearing  agent. 
After  clearing,  xylol  balsam  and  a  thin  cover  are  to  be  applied.  Carbol-thionine  (/) 
is  an  excellent  stain  for  general  use  with  sections,  alcohol  being  used  as  the  decolorizing 
reagent ;  Gram's  method  and  the  Ziehl  carbol-fuchsin  (with  Gabbet's  solution,  if  desired) 
are  also  entirely  appropriate.  As  a  counterstain  in  Gram's  method  borax-carmine 


208  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

may  be  employed  before  staining  with  the  aniline-water-gentian-violet  solution;  or 
hematoxylin  may  be  used  after  aniline-water-fuchsin  solution. 

The  above  description  of  staining  methods  and  the  necessary  reagents  is  neces- 
sarily brief,  reference  being  made  to  but  very  few  of  the  many  valuable  methods  appli- 
cable to  general  or  special  use.  For  more  detail  and  a  more  complete  list  of  the  methods 
proposed  reference  should  be  made  to  the  standard  text-books  and  systematic  works 
on  bacteriology.  The  special  methods  of  staining  spores,  flagella,  etc.,  are  given  in 
connection  with  the  paragraphs  devoted  to  such  bacterial  structures. 

PHYSICAL  CHARACTERISTICS  OF  BACTERIA. 

i.  Shape  of  Bacteria. — Bacteria  are  single-celled  organisms.  In  the  unstained 
preparations,  or  better  after  staining,  they  are  seen  to  present  certain  shapes,  which 
are  in  the  main  characteristic.  Uniform  persistence  of  such  shape  or  its  variability 
under  altered  conditions  of  growth  constitutes  an  important  feature  in  the  oligo- 
morphism  and  pleomorphism  of  bacteria.  The  oligomorphous  varieties  (eubacteria] 
are  by  far  the  most  numerous  and  include  all  the  known  types  important  in  medicine ; 
the  pleomorphous,  exhibiting  a  wide  possibility  of  adaptation  and  growth,  are  essen- 
tially saprophytic  and  numerically  limited  and  of  little  except  general  interest.  It 
is  not  meant  that  the  shape  of  a  given  oligomorphous  bacterium  is  absolutely  fixed 
and  invariable,  however;  certain  variations  in  shape  and  size  being  essential  in  the 
growth  of  these  as  of  any  germs,  and  some  minor  modifications  in  both  of  these  features 
and  in  the  physiologic  phenomena  being  manifest  from  the  influence  of  special  condi- 
tions of  development  (evolutional  and  involutional  forms) .  However,  such  variations 
are  not  of  such  degree  as  to  change  the  essential  type.  Three  morphologic  major 
types  may  be  distinguished — the  globular  or  short  oval  forms,  the  long  ovals  or  rods,  and 
the  curved  rods.  These  served  as  the  basis  for  the  older  classifications,  the  first  being 
known  as  cocci  (sing.,  coccus),  the  second  as  bacilli  (sing.,  bacillus),  and  the  third  as 
spirilla  (sing.,  spirillum) ;  and  these  terms  are  constantly  used  in  general  application 
where  better  definition  is  not  demanded.  The  modern  classifications,  while  based 
upon  the  general  outline  of  bacterial  shape,  include  other  features  and  are  much  more 
complicated  than  such  a  simple  division.  In  the  classification  of  Migula,  adopted 
with  minor  modifications  in  these  pages,  the  first  group  is  included  under  the  family 
name  Coccacece,  the  second  under  the  term  Bacteriaceae,  and  the  third  under  that  of 
Spirillacea.  The  presence  or  absence  of  organs  of  motility,  of  definite  capsules,  of 
branch-divisions  of  the  bacterial  cell,  the  mode  of  grouping,  and  a  number  of  other 
features  are  the  basis  for  further  division.  A  rod-shaped  organism  showing  true 
branching  is  spoken  of  as  a  mycobacterium;  the  claustridium  is  a  rod-shaped  germ  with 
a  thick,  clubbed  extremity  or  thick  in  the  middle  and  tapering  at  the  ends. 

These  shapes  can  usually,  when  marked,  be  distinguished  without  special  prepara- 
tion ;  but  for  their  clear  demonstration  they  are  best  prepared  by  one  or  other  of  the 
methods  of  staining. 

Exercise  41. — Prepare  two  films  of  cocci  from  a  known  culture  of  Micro- 
coccus  pyogenes  aureus,  taking  care  not  to  add  too  much  of  the  growth  to 
the  drop  of  water  used  in  making  the  film  lest  the  preparation  be  so  crowded 
that  observation  with  the  microscope  will  be  difficult.  Stain  one  film  with 
the  ordinary  watery  solution  of  gentian  violet,  without  heat,  for  four  or 
five  minutes.  Wash  well  with  water.  Stain  the  second  film  bv  Gram's 


210  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

method.  Dry  and  examine  in  oil  without  cover.  Note  the  shape  of  the 
individual  organisms;  and  after  comparison,  note  the  results  of  use  of 
Gram's  stain. 

Exercise  42. — From  fresh  gonorrheal  pus  repeat  the  above  exercise. 
Note  the  gonococci  lying  in  small  groups  in  the  cells  in  the  preparation. 
Note  shape  of  the  organisms.  Has  Gram's  method  been  successful? 

Exercise  43. — From  a  known  culture  of  Bacillus  typhosus  repeat  exer- 
cise 41.  Note  the  shape  of  the  organisms.  Has  Gram's  stain  been  suc- 
cessful ? 

Exercise  44. — From  a  known  culture  of  Microspira  comma  repeat  exer- 
cise 41,  using  as  the  staining  solution  for  the  first  film  a  dilute  (i :  10)  solu- 
tion of  carbol-fuchsin  for  five  or  ten  minutes  with  gentle  heat.  Note  the 
shape  of  the  organisms.  Does  Gram's  method  succeed? 

2.  Grouping  of  Bacteria. — The  occurrence  of  bacteria  in  peculiar  arrangement 
is  usually  the  result  of  incompleteness  of  separation  in  the  cellular  division  of  the 


& 


G 

FIG.  60. — GROUPING  OF  BACTERIA. 

A.   Micrococci.     B.    Diplococci.      C.    Streptococci.     D.    Sarcinae.     E.    Tetrads.     F.    Chains 
of  rods  (streptobacilli).      G.  Chains  of  curved  rods  (spirochaetse). 

ordinary  vegetative  mode  of  reproduction.  In  multiplication  in  this  manner  the  cells 
may  divide  in  one,  two,  or  three  planes.  In  the  first  case  the  individuals,  if  not  com- 
pletely parted,  remain  placed  end  to  end,  chains  or  filaments  being  thus  produced ; 
in  the  second,  division  occurring  laterally  and  longitudinally,  films  or  merismopediae 
(sing.,  merismopedia)  are  produced;  in  the  third,  division  taking  place  in  length, 
breadth,  and  thickness,  packet  forms  (sarcina)  or  massive,  irregular  groups  (zoogle&r 
sing.,  zooglea)  are  formed.  Such  grouping  may  be  often  seen  in  unstained  prepara- 
tions, as  in  the  hanging  drop,  but  best  in  well-stained  films.  In  staining  so  as  to 
make  clear  the  delicate  lines  of  division  between  the  individuals  of  such  groups,  it  is 
advisable,  after  having  fixed  the  film  by  heat,  to  immerse  it  for  a  minute  or  less  in  a 
weak  (four  per  cent.)  solution  of  acetic  acid.  The  best  stains  are  accomplished  by 
very  dilute  staining  solutions  left  in  contact  with  the  film  for  a  prolonged  period  (one 
of  ordinary  stain  to  four  of  distilled  water,  left  in  contact  with  the  film  for  ten  or  fifteen 


212 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


minutes).     Impression  films  here  find  their  particular  use,  and  should  in  any  doubtful 
case  be  resorted  to. 

From  peculiar  modes  of  grouping  which  are  more  or  less  characteristic,  these 
special  terms  are  commonly  applied:  When  cocci  occur  singly,  the  individuals  are 
spoken  of  as  micrococci  (a) ;  when  in  twos,  as  diplococci  (b) ;  when  in  chains,  as  strepto- 
cocci (c) ;  in  packets  of  eight  or  geometric  multiples,  as  sarcina  (d) ;  when  in  planes 
of  four,  tetrads  (e)  (Fig.  60).  The  term  staphylococcus  was  first  applied  to  cocci  whose 
gross  colonies  presented  a  fancied  resemblance  from  their  lobulated  outlines  to  a  bunch 
of  grapes;  later,  the  idea  of  small  microscopic  clusters  of  the  individual  cocci  became 
attached  to  the  name.  The  term  has  no  real  place  in  terminology,  the  same  idea 
being  conveyed  by  the  common  term  "elumps,"or  zooglea.  When  rod-shaped  forms  occur 
in  chains,  one  usually  uses  the  term  chains  or  threads  of  bacilli,  etc.,  although  occasionally 
the  prefix  "  strepto-"  is  applied  (e.  g.,  streptobacilli}.  The  term  spirochceta  is  applied 
where  a  single  long  spiral  or  spiral  chain  of  short  curved  elements  exists. 

Exercise  45. — Examine  preparations  of  exercise  42  carefully  for  apprecia- 
tion of  the  diplococcus  shape  of  the  gonorrheal  organism;  and  that  of 
exercise  44,  with  a  view  of  meeting  with  spirochaeta- 
like  chains  of  the  short  comma-shaped  individuals. 

Exercise  46. — Prepare  an  impression  film  from  a 
colony  of  Bacillus  subtilis,  and  a  second  film  of  the 
same  organism  by  the  ordinary  method.  Stain  each 
with  ordinary  aqueous  solution  of  fuchsin  or  gentian 
violet.  What  differences  are  apparent? 

Exercise  47. — From  some  sputum  from  a  chronic 
tuberculous  patient  with  pulmonary  cavities  prepare 
a  film,  and  stain  with  carbol-fuchsin  and  Gabbet's 
solution ;  and  examine  preparation  for  tetrads  (Micro- 
coccus  tetragenus) ;  afterward  examine  the  red  Myco- 
bacteria  tuberculosis  for  branching  forms. 


—A 


B 


FIG.  61.  — STRUC- 
TURE OF  A  BAC- 
TERIUM. —  (After 
Migula.} 

A.  Cell  membrane. 
B.  Protoplasm.  C. 
Vacuole.  D.  Me- 
tachromatic  gran- 
ules. 


3.  Structure  of  Bacteria. — The  bacterial  cell  is  composed 
of  a  cell  membrane,  a  layer  of  protoplasm,  and  a  central  fluid 
substance.  It  is  not  definitely  established  that  bacteria 

possess  nuclei,  but  after  proper  staining  there  are  likely  to  be  found  in  the  proto- 
plasm of  the  cells  certain  granules  which  by  some  are  regarded  as  of  nuclear 
character.  From  their  coloring  reactions  they  are  sometimes  spoken  of  as  meta- 
chromatic  granules.  They  are  not  ordinarily  seen.  The  cell  membrane  is  generally 
thin,  but  in  some  cases  it  is  thick  and  not  well  defined  on  the  outer  surface,  giving  the 
appearance  of  a  mucous  envelope  or  capsule  (whence  the  class  of  "capsule  bacteria"}, 
and  is  not  readily  and  clearly  stained  by  the  ordinary  methods.  Such  capsules,  more- 
over, are  not  apt  to  appear  in  bacteria  grown  in  the  ordinary  media,  but  are  best  seen 
when  the  organisms  are  obtained  directly  from  the  animal  body  or  when  grown  in 
fluid  blood-serum  or  a  few  other  special  laboratory  media.  The  term  ascococci  (sing., 
ascococcus}  is  applied  to  the  globular  forms  presenting  such  encapsulation;  in  other 
forms  the  name  capsule  bacteria  is  usually  used  (Fig.  62). 

The  outer  surface  of  the  cell  membrane  in  the  majority  of  bacteria,  especially  in 


214 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


the  globular  and  short  rod  forms,  is  devoid  of  appendages;  but  frequently  in  the  longer 
rods,  both  straight  and  curved,  and  occasionally  in  the  former,  there  are  to  be  found 
delicate  hair-like  or  lash-like  extensions  known  as  flagella,  regarded  as  the  means  of 
active  movement.     These  flagella  are  to  be  seen  after  the  application  of  special  methods 
of  staining,  best  in  young  cultures  grown  upon  solid  media;  and  are  by  no  means  con- 
stantly demonstrable  even  in  the  same  variety  or  in  all  forms  of  motile  bacteria  by  the 
methods  at  present  employed.      However,  they  are  sufficiently 
constant   to   be   of   service    in  classification  and   identification 
(Migula).     When  such  flagellum  is  single  upon  the  cell,  flagella- 
tion is  said  to  be  monotrichous ;  when  numerous  at  one  or  both 
poles  of  the  cell,  it  is  spoken  of  as  lophotrichous  (flagella  polar) ; 
when  single  at  each  pole,  amphitrichous ;  when  found  all  along 
the  sides  as  well  as  at  the  ends  the  flagellation  is  said  to  be  peri- 
trichous.     In  the  adopted  classification  coccus  forms  possessed 

of  flagella  are  termed  planococci  (sing.,  planococcus) ;  flagellated  sarcinae,  as  plano- 
sarcince  (sing.,  planosarcina) ;  a  straight,  rod-shaped  organism  with  polar  flagella,  as 
a  pseudomonas  (pi.,  pseudomonades  or  pseudomonads) ;  straight  rods  with  peritrichous 
flagella,  as  bacilli;  straight  rods  devoid  of  flagella,  as  bacteria;  comma-shaped  rods 


FIG.  62. — TYPES  OF 
CAPSULE  BAC- 
TERIA. 


D  £ 

FIG.  63. — FLAGELLATION  OF  BACTERIA. 
A.   Planococci.     B.    Pseudomonads.      C.   Bacilli.     D.  Microspine.     E.  Spirilla. 

with  one  or  two  lophotrichous  flagella,  as  microspirce  (sing.,  microspira) ;  comma- 
shaped  rods  without  flagella,  as  spirosomata  (sing.,  spirosoma) ;  the  term  spirillum 
is  retained  for  those  comma-shaped  rods  which  have  a  bunch  of  flagella  at  one  or 
both  poles  (Fig.  63). 

Exercise  48. — Staining  of  Supposed  Nuclear  Granules. — Make  film 
from  known  culture  of  Bacillus  coli  in  ordinary  manner;  but  in  fixing  pass 
through  the  flame  ten  times  instead  of  two  or  three.  Stain  four  or  five 
minutes  with  Loefrler's  solution,  boiling,  replacing  the  solution  on  the  film 
as  it  evaporates.  Wash  well  with  water  and  dry.  Examine  in  oil  without 
cover. 

Exercise  49. — Staining  capsules  (Johne). — Prepare  a  film  of  sputum 
from  a  case  of  acute  pneumonia,  as  before  described  for  sputum  from  a 
tuberculous  case.  Cover  preparation  with  two  per  cent,  aqueous  solution 


216  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

of  gentian  violet  and  warm  until  it  steams.  Wash  well  in  water;  moisten 
with  two  per  cent,  acetic  acid  solution  for  ten  seconds;  wash  in  water; 
cover,  and  examine  in  water  with  one-seventh  inch  dry  lens.  If  not  suc- 
cessful, remove  cover  and  repeat.  If  pneumonic  sputum  be  not  on  hand, 
use  tubercular  sputum  containing  Micrococcus  tetragenus,  which  also  pre- 
sents a  well-marked  capsule. 

Exercise  50. — Staining  Flagella  (Lowit). — Have  five  slides  perfectly 
free  from  fat  (first  washing  with  soap  and  water,  rinsing  in  water,  then  in 
alcohol  and  ether,  drying  with  paper;  fluid  should  spread  evenly  and  re- 
main in  a  thin  film,  not  in  drops,  if  slides  be  clean).  Use  young  cultures, 
twenty  to  twenty-four  hours  old,  on  agar,  of  the  following  organisms: 
Planococcus  agilis,  Pseudomonas  pyocyanea,  Bacillus  typhosus,  Bacillus  coli 
and  Microspira  comma,  for  the  preparation  of  films.  On  each  slide  place 
three  small  drops  of  distilled  water.  With  usual  precautions  take  from  the 
first  culture  a  bit  of  the  surface  of  the  growth,  and  with  a  single,  circular, 
sweeping  movement  diffuse  some  of  the  bacteria  in  the  first  drop  of  the 
water  on  one  of  the  prepared  slides.  Sterilize  needle  in  flame.  Take  a 
little  of  this  first  drop  and  in  a  similar  manner  diffuse  in  the  second.  Flame 
needle.  Repeat  diffusion  from  second  to  third  drop.  Dry  all  three  drops 
in  the  air  and  fix  films  by  passing  slide  through  the  flame  only  once  or  twice, 
taking  care  not  to  overheat.  In  the  same  manner  prepare  and  fix  films 
from  each  of  the  remaining  cultures  on  the  other  slides,  and  in  turn  stain 
each  preparation  as  follows :  Apply  the  following  freshly  prepared  mordant, 
allowing  it  to  act  for  two  or  three  minutes,  cold. 

Tan.nic  acid, 5 

Distilled  water 20 

Dissolve  and  filter  twice.  To  ten  cubic  centimeters  of  the  solution  add  five  cubic  centi- 
meters of  a  saturated  aqueous  solution  of  copper  sulphate,  well  filtered,  and  one  cubic  centi- 
meter of  a  saturated  alcoholic  solution  of  fuchsin.  Filter  the  mixture  twice. 

After  action  of  this  mordant,  wash  preparation  well  in  water;  there- 
after stain  with  aniline- water-gentian-violet  for  five  minutes,  cold.  Wash 
well  in  water  and  for  one  or  two  seconds  in  fifty  per  cent,  alcohol.  Dry  and 
examine  as  usual.  If  unsuccessful,  repeat.  Note  the  different  types  of 
flagellation  presented  by  these  organisms.  Does  Bacillus  typhosus  or  Ba- 
cillus coli  exhibit  the  greater  number  of  peritrichous  flagella  ? 

4.  Size  of  Bacteria. — Individual  bacteria  of  the  same  kind  are  fairly  uniform  in 
size,  a  feature  of  some  importance  in  identification.  One  must  recognize,  however,  a 
certain  variation  as  occurring  in  case  of  the  same  microorganism,  particularly  when 
grown  under  different  conditions  and  at  the  beginning  of  growth  as  well  as  in  old 
colonies.  The  statement  of  size  must,  therefore,  refer  to  average  individuals,  or  must 


15 


218  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

express  the  extremes  of  variation.  In  making  measurements  one  should  not  be  content 
with  but  a  single  observation,  but  should  measure  a  number  of  individuals  of  the 
preparation  (and,  too,  of  the  same  organism  grown  under  varying  conditions),  express- 
ing as  the  result  of  the  investigation  either  the  range  of  size  or  the  average  size  of  the 
individuals. 

For  measurement  of  such  objects  the  same  technique  is  to  be  employed  as  in 
measuring  other  minute  bodies.  A  reliable  stage  micrometer  is  focussed  with  the 
various  lenses  (no  oil  to  be  used  when  employing  the  oil-immersion  lens),  and  when 
proper  focus  is  obtained  for  each,  an  eyepiece  fitted  with  micrometer  substituted  for 
ordinary  ocular.  The  divisions  of  the  eyepiece  micrometer  are  now  calculated  in 
relation  with  the  known  measurements  of  the  stage  micrometer  and  carefully  recorded 
lest  they  be  forgotten.  The  stage  micrometer  is  now  put  aside  and  the  microscopic 
preparations  of  the  bacterium  substituted  and  brought  into  clear  focus,  the  length 
and  thickness  for  at  least  ten  individuals  determined  by  comparison  with  the  rulings 
in  the  eyepiece  micrometer,  and  the  limits  or  the  average  set  down  as  the  result.  Thus, 
one  would  express  the  measurements  of  Bacillus  typhosus  as  0.5-0.8  :  1-3  //,  meaning 
that  it  is  from  five-tenths  to  «ight-tenths  of  a  micromillimeter  in  thickness  and  from 
one  to  three  micromillimeters  in  length  (micromillimeter,  one  thousandth  of  a  milli- 
meter, one  twenty-five-thousandth  of  an  inch).  The  ordinary  bacteria  range  from 
somewhat  less  than  one  micromillimeter  to  five  or  ten  micromillimeters  in  long  diameter, 
but  there  are  organisms  much  smaller,  and  others  three  or  four  times  the  larger  limit 
just  mentioned. 

Exercise  5 1 . — Calculate  the  value  of  the  spaces  in  the  eyepiece  microm- 
eter for  the  one-twelfth  inch  oil-immersion  lens,  and  for  the  one-seventh 
inch  dry  lens.  Then  determine,  as  instructed,  the  size  of  the  bacteria  of 
several  of  the  previously  stained  preparations  (e.g.,  Micrococcus  pyogenes 
aureus;  Bacillus  typhosus;  Mycobacterium  diphtheria;  Mycobacterium 
tuberculosis} . 

5.  Motility  of  Bacteria. — One  of  the  most  striking  phenomena  to  be  noted  in 
examining  unstained  specimens  of  bacteria  suspended  in  some  fluid,  as  in  a  hanging 
drop,  is  the  motion  exhibited  by  the  individual  cells  of  many  types.  (This  power  of 
movement  may  be  so  persistent  that  it  may  remain  for  some  minutes  in  a  dried  but 
poorly  fixed  film,  even  after  staining  and  mounting  in  balsam.)  The  movements  may 
be  very  active,  darting  or  undulating  in  type;  or  may  be  slow  and  scarcely  perceptible. 
One  must  not,  however,  confuse  the  quivering,  dancing  movement  ("Brownian  move- 
ment") which  may  appear  in  case  of  non-motile  bacteria,  as  well  as  in  case  of  any 
inert  or  dead  particles  when  suspended  in  a  fluid,  for  true  movement;  when  difficulty 
is  met  in  distinguishing,  one  may  place  the  organism  in  some  germicidal  solution,  as 
of  carbolic  acid  or  corrosive  sublimate,  when  if  movement  persist  it  is  not  due  to  organic 
Energy.  The  passive  motion  induced  by  currents  under  the  cover-glass,  bearing  the 
bacteria,  motile  and  non-motile,  in  streams  in  different  directions,  must  not  be  mistaken 
for  active  motion.  The  means  of  these  active  progressive  movements  were  long  sus- 
pected to  be  due  to  the  possession  of  ciliate  or  flagellate  organs  before  the  discovery 
of  such  appendages  by  special  methods  of  demonstration.  Even  to-day  it  is  impossible 
from  insufficiency  of  our  methods,  however,  to  recognize  these  organs  in  a  number  of 
motile  varieties.  In  the  family  of  the  Beggiatoaceae  a  peculiar  creeping  or  waving 


220  LABORATORY  EXERCISES  IN  BACTERIOLOGY, 

movement  is  noticed,  supposed  to  be  due  to  the  presence  of  an  undulating  membrane 
attached  to  the  organism,  as  in  case  of  the  oscillatoria. 

To  this  power  of  movement  must  in  part  be  referred  the  active  approach  or  retreat 
of  motile  bacteria  to  foci  of  various  substances,  as  points  of  disease  in  the  body  (chemo- 
taxis) ;  the  similar  change  of  location  of  non-motile  forms  probably  depending  upon 
growth-progression  or  passive  convection  by  currents  in  the  surrounding  fluids. 

Exercise  52. — Rub  up  a  little  carmine  with  distilled  water.  Arrange  a 
drop  as  a  hanging  drop,  and  examine,  noting  the  dancing  movement  of  the 
grains.  Arrange  similarly  a  drop  of  water,  after  diffusing  in  it  a  few  bac- 
teria from  a  growth  of  Micrococcus  pyogenes.  Has  it  a  similar  movement? 
With  a  needle  touch  a  tiny  drop  of  carbolic  acid  to  the  drop  and  again  ex- 
amine. Does  it  continue  to  move  ?  What  is  the  source  of  movement  ? 

In  the  same  way  prepare  a  hanging  drop  of  Bacillus  typhosus.  Note 
the  movement  of  the  individual  rods.  How  does  it  differ  from  that  in  the 

previous  preparation?     Add   again  a    trace  of 

A  o  G>  co  ec  carbolic  acid.     What  effect  has  it  had  upon  the 

an  rr>  a\  CD  o&  oc  movement? 

£)W  VJ   t£>   g£j  Tljr     VQ 

e  Exercise  53. — In  a  similar  hanging-drop  pre- 

paration of  Bacillus  typhosus  suspend  the  smallest 
possible  fragment  of   sterile,  solid  agar.     Note 
for  fifteen  to  thirty  minutes  the  approach  and 
FIG.  64.— REPRODUCTION  OF    attachment  of   the   bacilli  to  the  fragment  as 
A.  Division^coJc'us  into  dip-    illustrative  of  positive  chemotaxis.     (Can  there 
lococci  or  two  cocci.     B.     be  any  relation  between  this  experiment  and 
Ct0Intorasardoaf0or  Tghi    the  agglutination  phenomenon  of  the  organism?) 

cocci.     D.  Division  of  rod 

form   by   transverse  separa-  6-  Reproduction    of    Bacteria.— Under    ordinary 

tion  into  two  rods.  circumstances  the  majority  of  bacteria  multiply  as  far  as 

is  known  by  direct  cell  division,  a  delicate  extension  of  the 

cell -wall  extending  from  the  sides  through  the  cell-body  in  one,  two,  or  all  three  planes. 
The  globular  forms  may  divide  in  all  three  planes,  but  the  rod  forms  are  limited  to 
a  single,  lateral,  division.  Thus  a  coccus,  before  dividing,  becomes  slightly  elongated 
(Fig.  64)  into  an  oval;  and  the  division  occurring  in  one  direction,  it  is  separated  into 
two.  These  two  may  not  be  entirely  parted,  and  remaining  together  constitute  a 
diplococcus.  If  in  the  subsequent  division  of  these  and  their  offspring  in  the  same 
manner  the  separation  of  the  individuals  should  not  be  complete,  a  chain  of  strepto- 
cocci results.  Sometimes  after  division  in  one  direction,  or  coincidently  with  it,  a 
second  division  occurs  at  right  angles  to  the  first  plane  of  division,  whence  four  indi- 
viduals, or  two  diplococci,  or  one  tetrad,  may  result.  Or,  again,  a  third  division  at 
right  angles  to  the  other  two  may  take  place,  whence  a  sarcina  form  or  a  zooglea 
of  separate  individuals  may  arise.  It  is  estimated  that  complete  division  of  Micro- 
spira  comma  (Asiatic  cholera)  requires  twenty  minutes,  leading  to  the  possibility  of 
billions  of  individuals  within  a  single  twenty-four  hours  from  an  original  organism; 
and  it  may  be  said  that  under  fair  conditions  of  growth  most  bacteria  will  divide  within 
thirty  or  forty  minutes. 


222  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

Some  bacteria  frequently,  under  comparatively  normal  circumstances,  and  many 
forms  under  conditions  of  difficult  life  (as  from  unfavorable  amount  or  quality  of  food, 
moisture,  atmosphere,  temperature,  etc.),  may  produce  within  their  substance  certain 
highly  refractive,  definite  bodies  known  as  spores,  which  retain  the  vital  power  of  the 
bacteria  under  adverse  states  for  a  long  period;  and  from  which  subsequently  new 
bacteria  of  the  same  type  originate.  They  are  thus  analogous  to  the  spores  or  seed 
bodies  of  higher  types  of  fungi,  but  are  better  thought  of  as  really  only  resting  forms 
of  the  original  bacterial  bodies.  They  are  of  importance  in  the  study  of  the  different 
varieties  of  bacteria  for  identification  and  appreciation  of  qualities  of  vital  persistence. 

Such  spores,  while  probably  always  truly 
r—ip  A          within  the  bacterial  body  in  their  forma- 
Q  tion    (hence    endospores},   are   sometimes 

seen  free  (exospores}  in  the  midst  of  adult 

_    _  -  *2*o*         bacteria,  or  attached  to  the  ends  of  the 

E  f  9-  bacterial  cells  (arthros pores'),  the  envelope 

of  the  original  bacterial  cell  having  dis- 
FIG.  65.-TYPES  OF  SPORULATION.  appeared  from  about  the  spore      R  .g  a 

A,B,  C,  D,  and  ^Endospores.     ^and  G  .      fe  . 

Exospores.     A,  B,  D.   Spores  equatorial. 

Cand£.  Spores  polar.     F.  Arthrospores.         formed    from    one    bacterium;    from   the 

position  in  which  it  occurs  it  is  described 

as  equatorial  or  polar  (Fig.  65) ;  and  the  cell  outline  is  apt  therefrom  to  be  swollen 
into  claustridium  or  club  shape. 

For  the  development  of  these  spores  into  the  ordinary  vegetative  forms  there 
must  be  afforded  proper  conditions  of  life,  particularly  plenty  of  moisture,  through  the 
imbibition  of  which  apparently  the  bacterium  is  enabled  to  grow  and  force  its  mass 
through  the  capsule  of  the  spore.  This  is  spoken  of  as  germination  of  the  spore;  it 
may  take  place  at  one  or  both  poles  or  along  the  sides,  and  is  hence  described  as  polar, 
bipolar,  or  equatorial  germination  (Fig.  66).  Usually  spores  are  met  among  the  rod- 
shaped  organisms,  but  the  globular  forms  are  now  and  again  also  capable  of  spore 


, 


lj 


FIG.  66. — FORMS  OF  BACILLI  SHOWING  SPORES. 

formation.  They  are  often  easily  seen  in  ordinarily  stained  preparations  as  rounded, 
or  oval  bodies  within,  or  among,  or  apparently  on  the  ends  of  the  ordinary  bacteria, 
unstained  and  peculiarly  shining.  Their  refraction  is  especially  appreciated  when 
the  field  is  darkened  by  contraction  of  the  diaphragm  of  the  substage.  By  prolonged 
staining  with  the  application  of  considerable  heat  and  the  use  of  a  mordant. in  the 
staining  fluid,  they  may  be  colored;  after  which  they  are  usually  with  more  difficulty 
decolorized  than  the  ordinary  bacteria,  which  makes  possible  their  differentiation. 
The  following  method  has  been  found  very  satisfactory  for  their  demonstration  (modi- 
fied from  Hauser1) :  A  drop  of  water  is  placed  on  a  slide  and  in  it  diffused  a  small 
amount  of  a  sporulating  culture  of  some  organism.  The  drop  is  spread  and  nearly 


224  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

dried  in  the  air.  Before  it  is  quite  dry,  the  film  is  flooded  with  carbol-fuchsin  solution, 
which  is  heated  nearly  to  boiling  and  evaporated ;  the  slide  with  the  evaporated  stain 
being  passed  several  times  through  the  flame  to  fix  the  film.  More  of  the  stain  is 
now  added  and  boiled  for  five  minutes,  fresh  solution  being  furnished  as  evaporation 
takes  place.  At  the  end  of  this  time  the  excess  of  stain  is  poured  off  and  the  film 
well  washed  in  hot  water  until  the  color  is  discharged  from  the  organisms  (but  not  from 
the  spores).  This  usually  requires  several  minutes;  and  it  is  well  from  time  to  time 
to  drop  a  cover  on  the  wet  film  and  examine  the  preparation  with  the  one-seventh 
inch  lens  to  determine  when  to  refrain  from  further  decolorization.  This  accom- 
plished, the  film  is  stained  for  a  few  seconds  with  Loemer's  blue,  washed  in  cold  water, 
dried,  and  examined.  The  spores  will  be  found  red,  the  bacteria  blue. 

In  addition  to  the  possibility  of  determining  the  presence  of  spores  by  actual 
microscopic  observation,  it  may  be  inferred  that  they  are  present  (and  further  search 
in  microscopic  preparations  should  then  be  made)  if  after  exposing  a  culture  for  ten 
minutes  to  80°  C.  inoculations  made  from  it  upon  fresh  medium  are  followed  by  growth. 
In  this  the  adult  forms  of  bacteria  are  destroyed  by  the  heat  exposure  and  only  spores 
are  likely  to  persist  in  their  vital  condition,  further  growth  depending  upon  their 
germination. 

It  is  believed  that  a  third  mode  of  reproduction  occurs,  at  least  in  the  higher 
forms,  as  the  mycobacteria,  from  segmentation  of  the  rods  into  small  coccoid  bodies, 
analogous  to  the  gonidia  of  the  hyphomycetes,  and  spoken  of  as  such  in  this  connec- 
tion. They  are  small,  not  so  refractive  as  spores;  and,  like  spores,  they  are  believed 
to  be  very  resistant  to  adverse  conditions  of  life.  Under  favorable  conditions  they 
develop  by  simple  growth  into  the  adult  types. 

Exercise  54. — From  an  agar  culture  of  Bacillus  subtilis,  grown  in  the 
incubator  for  several  days,  one  can  usually  obtain  numerous  spores  or 
sporulating  individuals;  often  nearly  the  whole  preparation  seems  made 
up  of  these  to  the  exclusion  of  the  ordinary  rods.  Prepare  and  stain  a  film 
from  such  source  according  to  the  above  instruction.  What  is  the  position 
of  the  spores  in  the  bacilli  ? 

Exercise  55. — In  a  few  drops  of  sterile  bouillon  place  a  number  of  spores 
obtained  from  culture  used  in  previous  exercise,  and  keep  in  incubator. 
Every  ten  minutes  for  an  hour  or  more  prepare  films,  staining  them  quickly 
with  Loeflfler's  solution,  warm.  The  spores  will  be  unstained,  the  germinat- 
ing organisms  blue.  Is  germination  equatorial,  polar,  or  bipolar? 

CHEMICAL  ACTIVITIES  OF  BACTERIA. 

In  the  growth  of  bacteria,  both  in  the  living  host  and  upon  laboratory  media,  it 
is  well  known  that  important  metabolic  changes  proceed  in  the  bacterial  cells,  as  well 
as  chemical  alterations  in  the  medium  on  which  they  develop.  Little  is  known  of 
these  processes,  but  in  connection  with  them  a  number  of  phenomena,  as  production 
of  pigment,  light,  heat,  various  types  of  fermentation  and  putrefaction,  and  other 
manifestations,  take  place  along  with  the  growth,  multiplication,  and  death  of  the 
bacterial  cell.  The  demonstration  of  such  changes  is  in  a  large  measure  favored  or 
prevented  by  the  character  of  the  medium  upon  which  the  bacteria  are  grown;  and 


226  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

the  precise  relationship  of  any  bacterium  to  a  given  standard  culture  medium  must 
in  the  study  of  these  chemical  phenomena  be  recorded  to  give  any  observations  their 
full  value  for  the  classification  of  the  organism. 

1.  Pigment  Production. — Various  pigments,  probably  analogous  to  the  coloring 
principles  of  higher  plants,  are  developed  by  a  number  of  bacteria  (known  as  chromo- 
genic  bacteria).     These  coloring  substances  are  in  same  instances  probably  retained 
within  the  bacterial  body ;  in  others,  are  diffused  more  or  less  in  the  medium  of  growth. 
They  are  of  varied  hues — yellow,  red,  blue,  violet,  black,  green,  fluorescent  (reflecting 
colors  different  from  the  hue  of  the  culture  itself),  or  iridescent  (manifesting  a  play 
of  colors  as  of  a  rainbow).     Little  is  known  of  these  pigments  other  than  the  few  pecu: 
liarities  mentioned  and  their  solubility  in  different  reagents,   as  water,   alkaline  or 
acid  solutions,  carbon  disulphide,  alcohol,  ether  or  chloroform.     They  are  of  complex 
organic  composition,  iron  being  probably  a  more  or  less  important  factor  in  most, 
its  absolute  absence  from  the  medium  of  growth  being  usually  followed  by  failure 
of  chromogenesis.     They  are  best  seen  in  cultures  grown  in  media  containing  carbo- 
hydrates, as  potato.     In  the  ordinary  bacteria  the  coloring  matter  is  not  visible  in 
the  individual  cells  as  examined  with  the  microscope ;  there  are  a  few  instances,  ap- 
parently approaching  the  lower  algae,  in  which  a  slight  green  tint  is  to  be  noticed, 
perhaps  of  the  nature  of  chlorophyll ;  and  in  the  thiobacteria  and  rhodobacteria,  granules 
of  a  reddish  or  violet  color  are  found  (sulphur  or  bacterio-purpurin) . 

Note. — Here  should  be  demonstrated  a  number  of  the  common  types  of  chromogeris 
in  gross  colony. 

Exercise  56. — Grow  Bacillus  prodigiosus  upon  potato  at  room  tempera- 
ture, and  another  similar  culture  at  37°  C.  Note  the  red  color  of  the  first 
and  the  absence  of  color  from  the  second.  Likewise  plant  this  organism 
on  agar  free  from  sugar  and  carry  it  through  several  generations  to  observe 
loss  of  chromogenesis. 

Exercise  57. — Plant  Pseudomonas  pyocyanea  upon  two  tubes  of  agar. 
Close  one  of  these  tubes  with  rubber  stopper  (or  put  in  anaerobic  jar), 
leaving  the  other  protected  only  by  the  cotton  plug.  Grow  in  incubator 
and  compare  appearance  of  growth  at  close  of  each  twenty-four  hours  for 
several  days. 

Pour  into  the  second  tube,  showing  a  beautiful  blue-green  color  diffused 
through  the  agar,  a  little  chloroform ;  allow  it  to  stand  for  some  minutes 
and  observe  the  solution  in  the  fluid  of  the  blue  color  (pyocyaniri).  In  a 
potato  tube  of  the  same  organism  note  the  yellowish-brown  color  of  the 
culture.  Press  upon  the  surface  of  the  growth  with  the  sterilized  platinum 
needle,  and  observe  that  in  a  few  minutes  a  green  color  has  succeeded  the 
brown  (chameleon  reaction  of  Pseudomonas  pyocyanea},  later  fading  back  to 
the  original  brown  tint. 

2.  Photogenic  Power. — The  phenomenon  of  light  production  belongs  to  a  small 
group  of  bacteria  and  is  apparently  a  characteristic  of  the  vital  activities  of  the  germs 
rather  than  due  to  any  chemical  product  of  the  organisms.     The  light  is  of  course 
not  visible  in  the  lighted  room,  but  is  apparent  at  night  or  in  a  darkened  room ;  and 


228  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

is  of  a  pale,  phosphorescent  type.  Like  pigment  production,  it  is  interrupted  or  modified 
in  intensity  by  conditions  altering  the  vital  activities  of  the  bacteria,  as  temperature, 
atmospheric  relations,  and  various  chemical  agencies,  and  the  like.  Photogenic 
bacteria  are  apt  to  be  encountered  in  cultures  obtained  from  salt  water  or  salt  fish ; 
and  photogenesis  is  most  apparent  and  best  preserved  when  such  bacteria  are  grown 
in  media  rich  in  saline  elements  (as  nutrient  gelatine  made  from  an  infusion  of  salt 
fish  in  natural  or  artificial  sea-water,  with  addition  of  one  per  cent,  of  peptone,  one 
per  cent,  of  glycerine,  and  0.5  per  cent,  of  asparagin — Neumann  and  Lehmann). 

Exercise  58. — Let  the  instructor  here  demonstrate  in  the  dark-room  the 
light  production  of  one  of  the  phosphorescent  bacteria  grown  on  above 
medium. 

3.  Ferments. — By  fermentation  is  meant  the  splitting  up  of  complex  organic 
molecules  into  simpler  ones  through  the  agency  of  a  so-called  enzyme,  or  non-living 
ferment,  which  is  itself  not  destroyed  in  the  process  it  excites.  Such  enzymes  may 
operate  in  the  presence  of  various  substances  which  are  harmful  to  the  bacteria  them- 
selves, and  are  found  active  in  the  germ-free  filtrate  from  cultures;  and  are  therefore 
to  be  regarded  as  not  residing  in  the  bacterial  cells,  but  rather  as  a  product  of  elimina- 
tion from  the  cells. 

(a)  Proteolytic  (Albumin-dissolving)  Ferments. — These  are  present  in  the  cultures 
of  a  large  number  of  bacteria,  and  give  rise  to  the  common  phenomenon  of  liquefaction 
of  gelatine  media  (the  glue  in  gelatine)  and  blood-serum.  The  production  of  peptones 
or  propeptones  is  a  result  of  such  liquefaction.  It  may  be  demonstrated  by  a  thorough 
filtration  of  a  liquefied  gelatine  culture  through  a  porcelain  filter,  adding  to  ten  cubic 
centimeters  of  the  filtrate  5  grams  of  ammonium  sulphate  and  keeping  it  warm  for 
half  an  hour  (thus  precipitating  all  albumins  but  the  peptones  and  propeptones),  then 
refiltering  and  testing  the  latter  filtrate  with  an  alkaline  solution  of  cupric  sulphate, 
when  the  peptones  strike  with  the  reagent  a  violet  color. 

Exercise  59. — Plant  in  a  flask,  or  a  number  of  tubes,  of  gelatine  medium 
Bacillus  prodigiosus,  and  allow  the  material  to  become  completely  lique- 
fied. Then  through  a  porcelain  filter  sterilized  in  the  autoclave  separate 
the  bacteria  from  the  liquid  holding  soluble  albumins  in  solution.  Divide 
the  latter  into  two  parts.  To  one  part  (10  cubic  centimeters,  at  least)  add 
an  excess  of  ammonium  sulphate,  which  will  precipitate  all  of  the  albumins 
but  the  peptones  and  propeptones.  Refilter  and  test  the  filtrate  with 
Fehling's  solution  of  copper  sulphate  for  the  violet  reaction  of  peptones. 

Add  the  second  part  of  the  original  filtrate  (free  from  bacteria)  to  a 
fresh  tube  of  gelatine;  observe  that  in  the  course  of  twenty-four  to  forty  - 
eight  hours  liquefaction  of  the  gelatine  takes  place  from  the  action  of  the 
bacteria-free  enzyme. 

Plant  a  tube  of  gelatine  each  from  known  cultures  of  Micrococcus  py- 
ogenes,  Bacillus  coli,  Bacillus  typhosus,  Mycobacterium  diphtheria,  and 
Microspira  comma.  Note  the  results  as  to  liquefaction  in  each  case. 


230  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

(b)  Diastasic  Ferments. — A  number  of  bacteria  possess  the  power  of  converting 
starch  into  glucose  by  means  of  an  elaborated  diastasic  enzyme. 

Exercise  60. — Inoculate  several  tubes  of  sugar-free  bouillon  (or  peptone 
solution)  with  Bacillus  subtilis  and  incubate  for  eight  or  ten  days.  Prepare 
a  thin  starch  paste,  adding  two  per  cent,  of  thymol  (which  should  be  tested 
for  sugar  previously,  to  insure  the  absence 'of  the  latter),  and  mix  equal 
parts  of  the  culture  and  paste.  Put  aside  for  six  or  eight  hours  in  the  in- 
cubator and  then  test  with  Fehling's  solution  for  the  presence  of  glucose. 

(c)  Invertin  Ferments. — Such  ferments  are  developed  by  a  few  bacteria,  and  accom- 
plish the  transformation  of  cane-sugar  into  glucose. 

Exercise  61 . — Make  a  ten  days  old  culture  of  Microspira  comma  in  sugar- 
free  bouillon.  Prepare  a  two  per  cent,  solution  of  cane-sugar,  adding  two 
per  cent,  of  strong  carbolic  acid.  Mix  equal  parts  of  the  culture  and  sugar 
solution.  The  carbolic  acid  should  restrict  the  bacterial  activities,  but  will 
not  prevent  the  action  of  the  enzyme;  and  if  the  mixture  be  allowed  to 
stand  for  several  hours  and  then  tested  with  Fehling's  solution,  the  glucose 
reaction  will  be  obtained.  As  a  control,  test  in  the- same  manner  the  cane- 
sugar  and  carbolic  acid  solution,  noting  the  failure  of  the  copper  reduction. 

(d)  Rennet  Ferments. — The  phenomenon  of  milk  coagulation  may  be  dependent 
upon  the  lactic  acid  produced  from  the  milk-sugar  in  a  culture  of  a  given  organism, 
or  to  an  enzyme.     To  demonstrate  the  latter  a  culture  of  some  bacterium  incapable 
of  such  acid  formation  is  to  be  selected,  as  Bacillus  prodigiosus. 

Exercise  62. — A  culture  of  Bacillus  prodigiosus  upon  milk  is  prepared 
and  placed  in  the  incubator  for  twenty-four  hours,  when  without  change  in 
color  the  milk  will  be  found  solidly  coagulated.  What  has  been  the  influ- 
ence as  to  reaction  of  the  medium? 

Or,  a  sugar-free  bouillon  culture  of  the  organism  is  prepared  (ten  days 
old)  and  filtered  through  a  porcelain  filter,  equal  parts  of  the  filtrate  and  of 
a  milk  tube  mixed  and  placed  in  incubator  temperature  for  a  few  hours, 
when  the  same  result  is  reached. 

Plant  in  milk  from  known  cultures  of  Micrococcus  pyogenes,  Bacillus 
typhosus,  Bacillus  coli,  Mycobacterium  diphtheria,  and  Microspira  comma. 
Note  results  in  each  case. 

A  number  of  other  types  of  fermentation  are  commonly  met  with,  as  the  alcoholic 
fermentation  of  sugar,  that  of  the  production  of  acids,  as  lactic  acid  or  acetic  acid 
from  sugar  and  alcohol;  the  production  of  alkaline  substances,  as  ammonia,  from 
proteids  free  from  sugar  (urea-fermentation)  by  various  bacteria.  It  is  claimed  that 
free  acid  is  formed  only  in  sugar-containing  media;  and  upon  the  supposition  of  the 
early  splitting  of  the  small  amount  of  meat-sugar  in  ordinary  media  or  in  media  to 


232  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

which  sugar  in  small  proportions  has  been  added,  is  explained  the  frequently  observed 
phenomenon  of  an  early  acid  reaction  in  such  growths,  followed  later  and  overcome 
to  an  alkaline  reaction  by  the  alkaline  substances  produced  in  the  splitting  of  the 
nitrogenous  molecules  into  ammoniacal  products.  These  fermentations  are  probably 
also  due  to  enzymes.  It  seems  probable  that  in  the  ordinary  alcoholic  fermentation 
this  enzyme  is  retained  in  the  bodies  of  the  ferment  cells.  Such  an  enzyme  by  high 
pressure  has  been  obtained  from  yeast  cells  and  is  known  as  zymose;  and  it  seems 
likely  that  a  similar  or  identical  substance  exists  in  bacterial  cells  capable  of  the  same 
process.  In  effect,  in  such  processes  the  fermentescible  substance  probably  goes  through 
the  bacterial  body  and  the  process  is  closely  related  to  metabolism.  It  probably  is 
performed  for  acquirement  of  some  of  the  energy  (heat)  set  free  in  the  splitting  process. 
Closely  related  to  these  last  considerations  is  the  power  of 

4.  Gas  Production. — A  number  of  gases  may  be  produced  by  various  bacteria 
in  different  media.  In  the  sugar-containing  media  in  the  conversion  of  the 
sugar  molecule  into  alcohol  and  carbon  dioxide,  the  latter,  as  well  perhaps  as  a  few 
other  gases,  as  hydrogen  and  methane,  are  produced ;  and  in  the  albuminous  media 
ammoniacal  gases  and  sulphuretted  hydrogen  may  be  encountered.  For  the  purpose 
of  observation  of  gas  production  solid  media  may  be  utilized,  note  being  taken  of  gas 
bubbles  produced  in  the  media  which  have  been  inoculated  by  bacteria  capable  of 
generating  such  gases.  Or  liquid  media  in  the  fermentation  tubes  described  in  a  pre- 
vious section  may  be  employed. 

Exercise  63. — A  fermentation  tube  is  filled  without  bubbles  with  a 
bouillon  containing  one  per  cent,  of  glucose  or  other  sugar,  sterilized  in  the 
autoclave,  and  after  cooling  is  inoculated  with  Bacillus  coli  and  put  aside 
for  twenty-four  to  forty-eight  hours  in  the  incubator,  when  gas  will  be 
found  to  have  collected  in  the  closed  end  of  the  tube.  This  gas  is  largely 
composed  of  carbon  dioxide  from  sugar  destruction.  There  is  usually 
active  sulphuretted  hydrogen  formation  as  well.  To  recognize  the  latter, 
the  following  procedure  may  be  practised.  A  slip  of  paper  moistened  with 
a  solution  of  acetate  of  lead  is  fixed  in  the  bulb  of  the  tube  alongside  of  the 
cotton  plug  and  a  dark  rubber  cap  (free  from  sulphur)  tightly  fitted  over 
the  opening.  By  tilting  the  tube  the  gas  is  now  permitted  to  pass  to  the 
bulb,  and  after  allowing  it  to  stand  for  a  time,  the  lead  will  be  found  black- 
ened into  the  sulphide. 

If  now  in  a  similar  tube  the  amount  be  marked  by  wax-pencil  mark  on 
the  glass,  and  the  bulb  be  filled  with  a  ten  per  cent,  solution  of  sodium 
hydrate,  the  thumb  being  tightly  placed  over  the  opening  and  the  tube 
inverted  and  shaken  so  as  to  bring  the  soda  solution  and  the  gas  together, 
and  the  gas  thereafter  returned  to  the  closed  end  of  the  tube,  the  amount 
will  be  found  diminished.  This  loss  represents  the  proportionate  amount 
of  carbon  dioxide  which  was  present,  and  which  has  been  absorbed  by  the 
sodium  hydrate.  In  determining  the  amount  of  gas  generated  in  these 
tubes  proper  corrections  should  be  made  for  atmospheric  pressure  and 


16 


234  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

temperature;  or  at  least  the  conditions  of  pressure  and  temperature  noted 
in  the  records.  Moreover,  daily  records  should  be  kept,  and  the  amount 
accepted  as  final  only  four  or  five  days  after  gas  production  has  ceased. 
Repeat  with  Micrococcus  pyogenes,  Bacillus  subtilis,  Bacillus  typhosus, 
Microspira  comma,  and  Mycobacterium  diphtheria. 

5.  Acid  Production. — It  is  asserted  that  free  acids  are  produced  only  in  carbo- 
hydrate-holding media,  and  are  producing  in  sugar-holding    media  either  from  the 
sugar  directly  or  from  the  alcohol  after  alcoholic  fermentation.     Of  these  acids,  lactic 
acid  is  the  most  prominent,  but  there  may  also  be  traces  of  acetic  acid,  butyric  acid, 
and  a  few  others.     The  actual  analysis  of  the  media  for  such  acids  is  too  complicated 
for  discussion  in  this  place,  and  the  mere  demonstration  of  an  acid  change  in  the  reaction 
of  the  medium  must  be  sufficient. 

Exercise  64. — To  neutral  bouillon  is  added  a  drop  or  two  of  alcoholic 
solution  of  rosolic  acid,  so  as  to  produce  a  distinct  pink  color.  Inoculate 
the  tube  with  Bacillus  coli  and  place  in  the  incubator.  Note  that  for  a 
time  the  color  is  diminished,  indicating  the  production  of  an  acid  reaction ; 
after  a  few  days,  however,  owing  to  exhaustion  of  sugar,  this  ceases,  and 
from  the  production  of -ammoniacal  compounds  the  reaction  changes  to 
alkaline,  causing  the  color  to  reappear  and  grow  more  distinct. 

Repeat  with  cultures  of  Micrococcus  pyogenes,  Bacillus  subtilis,  Bacillus 
typhosus,  Mycobacterium  diphtheria,  and  Microspira  comma. 

Exercise  65. — To  sterilized  milk  a  little  sterile  litmus  solution  is  added 
so  as  to  cause  the  medium  to  become  slightly  blue.  To  such  preparation 
is  added  a  loopful  of  Bacillus  coli,  and  it  is  allowed  to  remain  in  the  incu- 
bator for  a  time.  At  first  the  blue  color  changes  to  red,  the  latter  later 
disappearing  and  a  deeper  blue  assumed  from  the  alkaline  change.  The 
milk-sugar  at  first  was  changed  to  lactic  acid ;  when  the  sugar  was  used  up, 
the  proteids  were  progressively  changed  into  alkaline  compounds. 

6.  Alkaline  Production. — This,  as  already  referred  to,  is   believed  to   be   due 
to  the  splitting  of  nitrogen  compounds  in  the  medium  employed  by  certain  bacteria 
in  a  manner  of  the  same  type  if  not  identical  to  metabolism;  and  the  products  as 
far  as  known  are  ammonia,  amine,  and  ammonium   bases.     It  is  the  same  process 
as  is  concerned  in  the  so-called  urea-fermentation,  by  which  the  urea  of  fresh  urine 
is  converted  into  ammonium   carbonate,   from   which  other  ammonium   bases  may 
thereafter  develop,  as  ammonium  urate  or  phosphate.     To  exhibit  this  alkaline  -pro- 
duction, which  is  common  to  a  large  number  of  bacteria  grown  on  sugar-free  proteids, 
the  following  will  serve: 

Exercise  66. — Bacillus  coli  is  inoculated  into  a  tube  of  peptone  solution 
and  grown  at  body  temperature  or  lower  (20  to  37°  C.),  for  from  ten  to 
fourteen  days.  The  medium  should  have  been  exactly  neutral  to  phenol- 
phthalein,  or  if  this  has  not  been  used,  a  fresh  tube  of  the  same  reaction  as 


236  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

that  employed  used  as  a  control.  A  drop  of  one  per  cent,  alcoholic  solu- 
tion of  phenolphthalein  should  be  used  as  an  indicator,  which  when  added 
to  the  inoculated  tube  at  once  strikes  the  usual  red  color  of  an  alkali.  If 
the  medium  was  not  exactly  neutral  to  phenolphthalein  when  the  organism 
was  introduced,  the  same  amount  of  the  indicator  should  be  added  to  the 
control  tube  and  the  intensity  of  color  in  the  former  compared  with  that 
in  the  latter  tube. 

Repeat  with  cultures  of  Micrococcus  pyogenes,  Bacillus  subtilis,  Bacillus 
typhosus,  Mycobacterium  diphtheria,  and  Microspira  comma. 

7.  Alkaloidal   Products. — Among   the   products   of   bacterial   activity   there   is 
a  large  group  (a)  of  basic  nitrogenous  bodies,  known  collectively  as  ptomaines,  belonging 
to  the  amines  (U"~HCH3)'  as  cadaverine  (N(CH2)2C2H5,OH),  sepsine  and  putrescine; 
ammonium  bases,  as  choline,  neuridine,  muscarine,  etc. ;  pyridine  derivatives  (derived 
from   pyridine,   C5H5N),   as  collidine   (C8HnN),   indol   (C8H7N),   and  skatol  (C9H9N) ; 
as  well  as  amido-acids  (as  leucine,  tyrosine,  etc.),  and  others. 

The  method  of  isolation  of  these  bodies  is  for  the  most  part  too  complex  for 
the  limits  of  the  present  outline.  Generally  speaking,  these  are  not  the  seriously 
poisonous  products  of  bacteria. 

(6)  Toxalbumins. — In  this  group  occur  a  number  of  the  essential  toxins  or  poisons 
of  pathogenic  bacteria,  bacterioproteins  (as  the  old  form  of  tuberculin  and  mallein), 
and  bacteria plasmins  (poisonous  principles  extracted  from  a  number  of  pathogenic 
organisms,  as  of  cholera  or  typhoid  fever,  by  pressure).  The  toxins  or  toxalbumins 
proper  are  substances  which  may  be  precipitated  as  amorphous  poisons  from  bouillon 
cultures  of  various  bacteria  (as  Mycobacterium  diphtheria)  by  the  ordinary  albumin- 
precipitating  reagents,  as  by  alcohol.  These  substances  are  extremely  unstable  and 
easily  destroyed  by  heat,  chemicals,  exposure  to  light,  and  other  agencies.  They 
are  the  most  important,  from  a  pathogenic  standpoint,  of  all  the  bacterial  poisons 
and  are  responsible  for  many  of  the  specific  symptoms  of  infectious  diseases;  and  in 
most  cases  are  active  in  extremely  small  amounts.  There  is  some  belief  that  instead 
of  true  albumins  in  these  bodies,  we  are  dealing  only  with  certain  unrecognized  bodies 
in  the  midst  of  the  albumins  of  the  medium  carried  down  in  precipitation. 

Bacterioproteins  differ  from  these  latter  in  that,  aside  from  their  smaller  toxic 
power,  they  are  not  altered  by  heat. 

8.  Antitoxins. — In  this  general  connection  should  be  mentioned,  briefly  because 
of  our  meager  knowledge  of  their  nature,  the  protective  substances  which  exist  or  are 
developed  in  the  fluids  and  tissues  of  the  animal  body  influenced  by  an  infection. 
These,  of  course,  are  not  substances  to  be  regarded  as  direct  bacterial  products,  but 
for  convenience  may  be  considered   here  in  outline.     The  protection  or  immunity 
of  an  individual  from  infections  depends  either  upon  some   mechanical  protection 
(as  that  of  the  skin  and  other  protective  coverings,  or  of  phagocytosis)  or  upon  certain 
chemical  antagonisms  (as  the  acids  or  alkalies  in  the  secretions  with  which  the  infection 
is  brought  into  relation,  or  the  alexins  and  antitoxins).     Alexins  are  protective  sub- 
stances naturally  pre-existing  in  the  fluids  of  the  body  (serum  and  lymph),  supposed 
to  be  derived  from  the  leucocytes.     They  have  not  been  isolated,  but  are  known  to 
be  very  unstable  to  heat  (55°  C.  or  above)  and  sunlight.       They  possibly  aid  phago- 
cytosis by  killing  bacteria,  after  which  the  leucocytes   may  more   efficiently   destroy 


238  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

and  remove  them.  Antitoxins  are  substances  which  are  developed  mainly  in  the 
blood-serum  during  the  course  of  an  infection,  which  have  antagonistic  influences  upon 
the  toxins  of  disease  (either  passively,  as  when  before  the  introduction  of  a  toxin  into 
an  animal  body  it  may  be  uniformly  rendered  innocuous  by  mixture  with  serum 
containing  the  antitoxin,  in  which  action  the  latter  may  be  thought  of  as  distinctly 
antidotal  and  protective ;  or  actively,  where  after  the  introduction  of  an  infection  into 
the  animal  its  effects  are  overcome  by  introduction  of  a  certain  amount  of  the  anti- 
toxic serum,  although  a  mixture  of  the  same  proportions  of  toxin  and  antitoxin  out- 
side the  body  and  its  introduction  into  a  smaller  animal  are  followed  by  the  usual 
effects  of  the  infection.  In  this  latter  form  the  antitoxin  apparently  stimulates 
some  elements  of  the  infected  body  to  a  direct  antagonism — or  is,  in  other  words, 
healing  in  its  influence). 

9.  Agglutination  Phenomenon.— In  this  same  connection  may  be  considered 
the  agglutinating  phenomenon  of  serums  of  persons  infected  or  recently  well  of  various 
diseases,  as  typhoid  fever,  over  the  bacteria  of  the  same  specific  disease.  This  mani- 
festation depends  upon  some  directly  bactericidal  .substance  (contrasting  with  the 
antitoxins  which  antagonize  rather  the  poisons  of  the  bacteria  in  the  serum) ;  in  some 
instances  actual  bacterial  destruction  (bacteriolysis}  may  take  place,  while  in  others 
rather  a  paralyzant  influence  is  exerted  upon  the  organisms  as  is  seen  in  the  stoppage 
of  movement  of  the  germs.  Where  such  bactericidal  substances  are  powerful  and 
persistent  in  the  serum  of  the  patient,  the  immunity  granted  to  the  individual  is  like- 
wise persistent.  The  nature  of  these  substances  is  not  understood. 

Exercise  67. — After  the  tenth  day  of  the  disease  in  typhoid  fever,  a 
finger  is  well  cleansed  and  dried  and  pricked  so  that  several  large  drops  of 
blood  may  escape.  These  are  caught  in  a  clean  glass  tube,  as  in  an  ordi- 
nary dropper  or  in  a  capillary  tube.  This  blood  is  allowed  to  clot  and  the 
serum  to  separate  (if  a  capillary  U-tube  is  used  it  may  be  centrifugated  for 
the  separation). 

A  twenty-four-hour  bouillon  culture  of  the  typhoid  bacillus  is  made. 
With  a  capillary  pipette  of  the  same  caliber  as  that  used  for  the  collection 
of  the  serum  a  suitable  amount  of  the  culture  is  withdrawn.  (Care  is  to  be 
taken  to  plug  the  upper  end  of  the  pipette  with  cotton  to  prevent  accidental 
suction  of  the  culture  into  the  mouth  of  the  operator.)  A  certain  length 
of  the  serum  tube  is  now  broken  off  (after  filing)  and  its  contents  blown 
out  into  a  watch-crystal  (one  centimeter  length  broken  off,  and  serum  ex- 
pelled by  holding  the  broken  part  in  forceps  and  blowing  it  out  with  another 
fine  tube  inserted  in  end) .  Fifty  times  this  amount  (ten  centimeters  length 
taken  five  times)  of  the  bouillon  culture  are  added  to  the  serum,  mixed, 
and  a  drop  placed  on  the  slide  and  covered  (hanging  drop  may  well  be 
made).  These  proportions  vary  with  different  observers,  as  weak  a  dilu- 
tion as  one  to  ten  having  been  originally  practised.  At  the  close  of  varying 
periods,  ranging  from  fifteen  minutes  to  as  much  as  two  hours,  according 
to  various  directions,  the  typhoid  bacilli  will  be  found  to  have  ceased  from 
active  movement  and  to  have  agglutinated  in  clumps.  Extreme  limits  of 


240  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

time  should  be  avoided  in  practice,  and  the  reaction  accounted  positive 
only  when  occurring  within  the  first  hour;  if  strong,  it  will  probably  have 
occurred  within  a  few  seconds.  Should  failure  occur  with  one  to  fifty 
dilution,  a  one  in  twenty-five  dilution  may  be  tried,  and  failures  with  this 
may  be  accounted  negative.  If  very  strong,  bacteriolysis  of  the  typhoid 
germs  may  also  be  noted,  the  destruction  beginning  with  vacuolization 
and  erosion  of  the  bacteria. 

The  same  result  may  often  be  obtained  in  a  tube  by  the  addition  of 
large  amounts  (in  same  proportions  as  above)  of  the  serum,  the  agglutin- 
ated masses  forming  a  flocculent  precipitate  visible  to  the  unaided  eye,  and 
the  previously  turbid  bouillon  becoming  clear  between  the  tiny  flakes.  In 
the  course  of  twenty -four  hours  the  effect  of  agglutination  and  stoppage  of 
motion  will  usually  be  found  to  have  disappeared,  the  agglutinating  agent 
having  apparently  been  exhausted  from  the  serum  employed. 

10.  Indol  and  Phenol  Production. — Among  the  ptomaines  indol  and  phenol 
are  substances  of  importance  in  that  they  are  demonstrable  with  little  difficulty, 
and,  being  more  or  less  characteristic  of  certain  organisms,  serve  to  aid  in  their  identi- 
fication. Aside  from  the  last  feature  it  is  not  known  that  these  substances  are  of 
importance. 

Indol  occurs  particularly  in  the  old  cultures  of  bacteria  of  the  colon  group  and 
of  the  various  microspirae  and  spirilla.  It  is  also  found  in  old  cultures  of  Mycobac- 
terium  diphtherice  and  bacterium  mallei.  It  is  shown  by  adding  to  a  tube  of  bouillon 
free  from  sugar  (or  the  ordinary  peptone  solution),  in  which  one  of  the  indol-producers 
has  been  grown  for  eight  to  ten  days,  an  equal  amount  of  weak  nitric  or  sulphuric 
acid  (twenty  per  cent.).  In  case  the  germ  has  also  produced  nitrites  in  the  medium, 
or  if  nitrites  be  present  in  the  nitric  acid  used,  the  reaction,  consisting  in  the  production 
of  a  red  color  (probably  due  to  the  formation  of  nitroso-indol  nitrate),  occurs.  But 
in  the  absence  of  the  nitrites  it  will  be  necessary  to  add  a  drop  of  a  one  per  cent,  solution 
of  sodium  or  other  nitrite.  Should  the  reaction  take  place  without  the  addition  of 
nitrite,  upon  the  addition  of  sulphuric  acid  alone,  it  may  be  tentatively  inferred  that 
the  organism  is  also  a  nitrite-producer. 

Exercise  68. — Grow  in  sugar-free  bouillon  or  peptone  solution  a  culture  of 
Bacillus  coli  for  eight  or  ten  days.  To  this  culture  add  an  equal  amount  of 
twenty  per  cent,  pure  sulphuric  acid,  and  if  needed  slightly  warm  for  a  few 
minutes.  Now  add  a  drop  of  a  one  per  cent,  solution  of  sodium  nitrite 
and  continue  to  add  and  warm  until  the  color  is  at  its  intensity.  Tco 
great  an  addition  of  nitrite  will  cause  a  yellowish  tint  to  be  assumed. 

Repeat  with  cultures  of  Micrococcus  pyogenes,  Bacillus  subtilis,  Bacillus 
typhosus,  and  Mycobacterium  diphtheria. 

Exercise  69. — To  a  similar  culture  of  the  cholera  organism  add  the  acid 
alone,  the  color  being  produced  without  the  addition  of  the  nitrite,  showing 
the  production  of  the  nitrite  as  well  as  of  indol  by  this  bacterium.  The 


242  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

reaction  has  been  long  known  in  connection  with  this  organism  as  the 
i( cholera  red  reaction." 

Phenol  production  occurs  with  much  the  same  groups  of  organisms  as  that  of 
indol.  For  its  demonstration  it  is  essential  to  use  a  larger  quantity  of  growth  in  sugar- 
free  bouillon  than  in  case  of  effort  to  demonstrate  indol  presence.  To  this  is  added 
one-fifth  its  volume  of  pure  hydrochloric  acid  and  the  mixture  distilled.  The  distillate 
contains  the  phenol,  which,  after  neutralization  of  the  distillate  with  calcium  carbonate, 
will  strike  a  violet  color  upon  the  addition  of  neutral  solution  of  chloride  of  iron  (one 
per  cent.). 

Exercise  70. — Make  a  culture  of  Bacillus  coli  in  sugar-free  bouillon  or 
peptone  solution,  using  about  fifty  cubic  centimeters  of  the  medium.  After 
eight  or  ten  days  add  ten  cubic  centimeters  of  hydrochloric  acid,  and  in  a 
condenser  (or  retort)  distil  five  or  ten  cubic  centimeters  from  the  mixture. 
Carefully  neutralize  with  powdered  calcium  carbonate  (or  calcined  magne- 
sia), and  add  drop  by  drop  a  one  per  cent,  solution  of  ferric  chloride  to  ob- 
tain the  violet  color  produced  by  phenol.  A  white  flocculent  precipitate 
may  be  produced  in  a  separate  part  of  the  distillate  upon  the  addition  of 
bromine. 

ii.  Nitrifying  and  Denitrifying  Bacteria. — Quite  a  large  number  of  bacteria 
apparently  possess  the  power  of  forming  nitrite  in  cultures,  although  it  is  not  clear 
how  this  is  accomplished;  and  possibly,  when  it  is  found  in  traces  only,  it  may  have 
been  absorbed  from  the  air.  In  the  study  of  indol  formation  the  lack  of  need  of  adding 
nitrite  to  bring  out  the  characteristic  reaction  in  many  examples  is  probable  evidence  of 
its  presence.  As  far  as  is  known,  certain  pseudompnads  found  in  the  soil  (Ps.  Europa, 
Ps.  Javanensis)  are  alone  able  to  convert,  the  first  the  nitrogen  of  ammonium  com- 
pounds into  nitrite,  the  second  nitrite  into  nitrate.  A  number  of  bacteria  found 
especially  about  the  roots  of  certain  plants  (e.  g.,  leguminosae)  apparently  absorb  the 
nitrogen  of  the  air,  preserving  it  in  the  soil  as  free  nitrogen  or  transforming  it  into  nitrites 
or  into  ammonium.  The  general  term  nitrifying  bacteria  is  applied  to  the  forms  en- 
gaged in  the  above  processes;  these  are  of  much  importance  to  the  agriculturist  in 
their  role  in  enrichment  of  the  soil. 

On  the  other  hand,  many  bacteria  possess  the  property  of  reducing  nitrates  to 
nitrites  by  the  abstraction  of  oxygen,  or  in  the  same  way  of  reducing  the  nitrites  to  free 
nitrogen,  sometimes  leading  to  the  formation  of  ammonium.  These  are  termed  the 
denitrifying  bacteria.  They  are  widely  distributed  in  dung,  soil,  and  putrefying  organic 
matter,  and  the  reduction  of  nitrates  to  nitrites  seems  common  to  the  majority  of 
bacteria.  The  reduction  of  nitrates  to  nitrites  is  usually  determined  by  growing  a 
bacterium  in  question  for  seven  days  either  in  the  room  or  incubator  temperature, 
as  is  best  fitted,  in  a  medium  made  up  of  water  with  but  a  small  amount  of  peptone 
added  and  with  a  little  saltpeter  (tap-water  1000,  1  gram  dried  peptone,  0.2  gram 
sodium  nitrate).  To  two  tubes  containing  about  three  cubic  centimeters,  each  thus 
prepared,  inoculated  and  grown,  and  to  a  control  tube  of  the  same  size  and  same  content 
of  the  saltpeter  broth  and  kept  in  the  same  conditions  as  the  inoculated  tubes,  is  added 
a  mixture  of  naphthylamine  and  sulphanilic  acid  (naphthylamine  1,  distilled  water 


244  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

1000 — kept  in  clean  glass-stoppered  bottle;  sulphanilic  acid  0.5  gram,  dilute  [1:16] 
acetic  acid  150  cubic  centimeters — also  kept  separate  in  glass-stoppered  bottle.  Mix 
equal  quantities  of  the  two  solutions  for  use.  Add  to  first  inoculated  tube  and  control 
tube  two  cubic  centimeters  each  of  the  mixture).  The  tubes  are  closed  with  rubber 
stoppers  and  allowed  to  stand  for  about  half  an  hour,  warming  hastening  the  reaction. 
If  nitrites  be  present  from  the  reduction  of  the  nitrate,  a  pink  or  red  color  develops, 
the  control  tube  remaining  colorless  or  perhaps  slightly  pink  if  it  has  absorbed  a  trace 
of  nitrite  from  the  atmosphere.  If  no  nitrites  are  thus  found,  no  reduction  may  have 
taken  place,  the  nitrate  remaining  unchanged  in  the  medium ;  or  the  reduction  may 
have  advanced  beyond  the  formation  of  nitrite  to  the  formation  of  nitrogen  or  ammonia 
To  determine  these  points  the  second  inoculated  tube  is  taken  up.  One-half  its  con- 
tents is  poured  into  a  clean  test-tube  and  a  strip  of  paper  wet  with  Nessler's  reagent 
suspended  over  the  medium,  when  if  fumes  of  ammoniacal  character  arise,  the  paper 
will  slowly  assume  a  yellowish  or  brownish-red  color.  The  remaining  half  of  the 
second  inoculated  medium  is  evaporated  to  dryness  either  in  the  tube  or  in  a  porcelain 
dish,  and  to  the  residue  is  added  a  drop  of  phenol-sulphonic  acid  (cone,  sulphuric 
acid,  c.  p.,  74  cubic  centimeters;  water,  6  cubic  centimeters;  and  pure  carbolic  acid, 
12  grams),  and  water  added  to  dilute  the  mixture  to  one  or  two  cubic  centimeters. 
This  is  then  alkalinized  by  addition  of  sodium  hydroxide  solution.  A  yellow  color 
indicates  the  persistence  of  the  nitrate  in  the  medium. 


LESSON  VIII. 

ISOLATION   OF   BACTERIA  IN   PURE   CULTURES. 

It  will  be  fortunate  if  after  inoculation  of  nutrient  substances  and  culture  of  the 
inoculated  media  the  organisms  grown  should  prove  from  their  gross  and  minute 
characteristics  to  be  of  a  single  type.  This  may  be  expected  only  in  instances  where 
the  efforts  of  the  operator  have  from  the  first  been  directed,  in  the  collection  of  the 
infected  material,  to  the  acquisition  of  but  a  single  species,  or  in  culture  to  permit 
conditions  favoring  the  development  of  but  a  single  type  of  organism;  in  the  best 
directed  attempts  it  is  not  uncommon,  and  in  ordinary  investigation  of  much  of  the 
material  likely  to  be  submitted  for  analysis  it  is  invariable  that  there  should  occur 
several,  perhaps  a  great  variety  of  growths  upon  the  media.  It  may  naturally  be 
.  inferred  as  impossible  to  study  with  any  success  the  characteristics  outlined  in  the 
preceding  two  chapters  for  any  bacterium  so  long  as  it  is  confused  with  others.  Hence 
arises  in  all  bacteriologic  study,  whether  of  pathogenic  or  non -pathogenic  forms,  the 
necessity  of  Koch's  second  postulate:  "The  organism  to  be  studied  must  be  obtained 
in  pure  culture." 

The  methods  available  for  the  isolation  of  organisms  in  pure  culture  arrange 
themselves  readily  into  two  groups:  (a)  mechanical  procedures,  and  (6)  methods  based 
upon  physiologic  peculiarities  of  the  bacterium  sought. 

(A)  MECHANICAL  METHODS. 

i.  Plating  Methods. — When  it  is  known  from  previous  experience  or  may  be 
reasonably  inferred  that  a  given  material  to  be  submitted  to,  bacteriologic  analysis 
contains  a  number  of  different  organisms  the  exact  nature  of  which  is  not  known 
(for  which  reason  it  is  impracticable  to  select  a  special  method  for  isolation),  efforts 
to  widely  distribute  the  organisms,  and  the  colonies  resulting  in  culture  from  their 
development,  over  an  extended  area,  offer  no  little  probability  of  success,  so  that  with 
the  sterilized  needle  each  growth  may  be  picked  up  and  transferred  to  a  separate 
container  of  sterile  medium  where  it  may  develop  alone.  Any  of  the  three  modes  of 
plating  may  be  employed, — plates,  Petri  dishes,  or  Esmarch's  tubes, — selection  of  one 
or  other  depending  mainly  upon  the  need  of  care  to  prevent  contamination,  but  also 
partly  upon  the  medium  of  growth  selected,  the  conditions  of  temperature  and  atmos- 
phere to  be  used  in  the  development  of  the  plated  culture,  and  upon  the  extent  of 
surface  believed  essential  for  proper  dissemination  of  the  bacteria.  One  would  not 
select  the  rolled  tubes  if  a  wide  surface  be  desired;  yet  they  offer  the  greatest  chance 
of  remaining  free  from  atmospheric  contamination  and  are  the  most  easily  manipulated 
in  incubator  and  anaerobic  conditions.  Plates  and  dishes  are  much  more  e  isily  made 
if  gelatine,  rather  than  agar,  be  the  medium  used.  Petri  dishes  are  less  liible  to  air 
contamination  than  plates,  and  are  more  conveniently  placed  in  the  incubator  or 
anaerobic  jar  and  more  readily  examined  in  the  course  of  the  culture.  In  any  com- 

246 


248  LABORATORY  EXERCISES  IX  BACTERIOLOGY. 

prehensive  work  it  should  not  be  neglected  to  arrange  not  one,  but  several  plated 
cultures  of  the  same  material  upon  agar  as  well  as  upon  gelatine,  with  a  view  of  placing 
one  culture  at  incubator  temperature,  one  at  room  temperature,  and  a  third  in  anaerobic 
surroundings.  Each  should  be  prepared  from  as  nearly  equivalent  original  material 
as  possible,  both  in  quantity  and  in  kind,  and  the  infected  material  thoroughly  diffused 
through  the  liquefied  medium  before  plating.  (One  must  not  forget  to  have  the 
liquefied  medium  of  as  low  a  temperature  as  possible,  lest  harm  be  done  to  the  bacteria 
diffused  in  it ;  and  after  diffusion,  when  about  to  pour  the  liquid  upon  a  plate  or  in 
a  dish,  let  it  be  kept  in  mind  to  flame  and  cool  the  lip  of  the  tube  for  fear  of  contam- 
inating the  medium  passing  over  it.)  In  addition  to  these  agar  and  gelatine  prepara- 
tions, smears  are  to  be  made  upon  solidified  blood-serum  and  submitted  to  incubator 
and  room  temperatures. 

After  proper  growth  has  been  attained  and  one  can  distinguish  one  type  of  colony 
from  another,  the  sterile  needle  is  to  be  used  for  transferring  some  of  the  organisms 
from  each  to  separate  tubes  of  sterile  medium,  identical  with  that  used  in  the  plated 
culture.  Care  is  to  be  exercised  in  this  step  that  the  tip  alone  of  the  needle  is  touched 
only  to  that  colony  from  which  transfer  is  to  be  made,  and  that  all  other  colonies  in 
the  preparation  are  avoided.  It  is  not  essential  to  have  visible  fragments  or  the 
entire  colony  on  the  needle.  It  will  be  found  of  advantage,  before  attempting  the 
procedure,  to  bend  the  tip  of  the  needle  at  right  angles  to  its  length,  this  shape  lending 
itself  to  the  operation.  After  the  needle  with  the  adhering  organisms  is  withdrawn 
from  the  culture,  it  is  introduced  to  the  tube  of  fresh  medium  and  a  stroke  or  stab 
inoculation  made,  after  which  the  needle  is  to  be  at  once  flamed.  Each  tube  thus 
inoculated  from  the  preliminary  culture  is  for  a  time  subjected  to  the  same  conditions 
as  those  which  proved  successful  for  the  plated  culture,  and  after  sufficient  develop- 
ment of  the  pure  cultures  the  colonies  in  each  are  noted  in  the  study  of  the  gross  and 
minute  features  of  the  organisms  composing  them. 

The  procedure  is  difficult  only  when  the  colonies  to  be  separated  are  very  close 
to  each  other  in  the  plated  culture;  and  care  in  manipulation  alone  will  then  insure 
success.  Should  one  of  the  tubes  inoculated  prove  to  be  mixed,  the  measure  is  to  be 
repeated  until  successful. 

Exercise  7 1 . — Several  loopfuls  of  Bacillus  prodigiosus  and  Micrococcus 
pyogenes  are  mixed  in  a  test-tube  containing  a  few  cubic  centimeters  of 
sterile  water.  Let  each  student  diffuse  a  loopful  of  the  fluid  in  liquefied 
gelatine  and  plate  in  one  or  other  manner,  subsequently  producing  from  the 
plate  a  pure  culture  of  each  organism  for  inspection. 

2.  Salomonsen's  Capillary  Tubes. — Salomonsen  suggested  that  after  diffusion 
of  the  infected  material  has  been  effected  in  a  sterile  liquefied  medium,  a  small  amount 
of  the  latter  be  drawn  into  a  sterile  capillary  glass  tube  instead  of  being  spread  out 
over  a  surface.  The  principle  is,  of  course,  the  same  as  in  any  of  the  plating  methods, 
the  germs  being  disseminated,  however,  in  a  linear  manner  instead  of  over  a  plane. 
After  the  tube  has  been  filled  with  the  inoculated  medium,  the  ends  may  be  sealed 
in  the  flame  or  closed  by  wrapping  a  little  sterilized  cotton  or  paper  closely  about 
them.  It  must  be  realized  that  in  such  tubes,  even  if  closed  in  the  latter  manner, 
but  little  air  access  is  permitted  to  the  organisms  in  the  medium  any  distance  from 
the  ends;  and  as  a  matter  of  fact  it  is  especially  in  the  isolation  of  anaerobic  varieties 


250  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

(facultative  and  obligate)  that  the  method  is  of  value.  It  is  the  practice  of  the  writer 
to  prepare  a  number  of  capillary  tubes,  inclosing  them  in  a  large  glass  tube  like  that 
used  in  Hesse's  air  apparatus,  both  ends  of  which  are  filled  with  cotton  plugs.  In 
this  they  are  sterilized  in  the  oven  and  kept  until  used.  When  filled  with  an  inoculated 
medium  and  closed,  each  capillary  is  marked  with  the  wax  pencil  so  that  it  may  be 
surely  distinguished  and  placed  for  culture  in  a  second  sterile  glass  tube  like  the  first 
in  order  to  prevent  the  outside  surface  of  the  capillary  from  contamination  and  to 
allow  observation  during  culture.  It  will  be  found  convenient  to  attach  to  this  con- 
taining tube  a  strip  of  card  or  paper  on  which  may  be  marked  the  time  and  appearance 
of  each  colony  for  each  of  the  contained  capillaries  as  it  appears,  as  is  indicated  in  the 
accompanying  diagram  (Fig.  67).  Several  capillaries  may  be  kept  in  the  enveloping 
tube  if  properly  marked  to  prevent  confusion.  When  growth  appears  in  such  a  capil- 
lary at  any  distance  from  the  ends,  whether  the  ends  have  been  sealed  or  not,  it  may 
be  accepted  as  either  a  facultative  or  obligate  anaerobic  colony;  when  the  tube  is 
sealed,  all  growth  appearing  is  anaerobic;  should  the  ends  not  be  sealed  and  growth 
occur  close  to  the  end,  it  may,  however,  be  an  aerobic  variety. 

When  it  is  desired  to  transfer  the  colonies  to  separate  culture  tubes,  a  capillary 
is  withdrawn  from  the  large  container  and  with  a  sterile  forceps  a  short  length  bearing 


*fi-/6-0?  white. round,  finely  granular,  margins  c/'//' 

_  -   ._  white    XT" 
ovo'd  glistening 


FIG.  67.— SALOMONSEN'S  CAPILLARY  TUBES  INCLOSED  IN  PROTECTIVE  GLASS  TUBE,  WITH 
LATTER  ATTACHED  TO  CARD  ON  WHICH  ARE  PRESERVED  NOTES  OF  COLONIES 
WITHIN. 

the  colony  broken  out  (one  end  of  the  fragment  should  be  very  close  to  the  colony) 
and  dropped  into  sterile  medium.  (It  is  well  first  to  transfer  to  bouillon  into  which 
diffusion  from  the  colony  in  the  capillary  fragment  readily  takes  place;  after  growth 
in  the  bouillon  one  or  two  loopfuls  are  smeared  over  the  surface,  or  a  stab  made  wdth 
the  straight  needle  into  the  interior  of  fresh  solid  medium.)  The  medium  inoculated 
with  the  colony  from  the  fragment  of  the  capillary  tube  should  for  several  days  be 
grown  in  the  anaerobic  jar,  its  growths  transplanted,  and  the  medium  then  exposed 
for  some  days  to  the  ordinary  atmosphere  in  order  to  afford  a  chance  of  development 
to  any  aerobic  individuals  which  possibly  may  be  present. 

This  method  is  especially  valuable  in  isolation  of  the  anaerobes,  facultative  or 
obligate,  in  studies  of  blood  or  other  pathologic  material  taken  from  some  diseased 
individual,  such  a  fluid  being  drawn  directly  into  the  capillary  tube  and  sealed  therein, 
where  it  serves  as  the  culture  medium  for  the  preliminary  growth  (McLaughlin). 

Exercise  72. — A  tube  of  liquefied  gelatine  is  inoculated  with  a  loopful 
each  of  a  bouillon  culture  of  Bacillus  typhosus  and  Pseudomonas  pyocyanea. 
Let  each  student  recover  in  pure  culture  and  present  for  inspection  the 
former  of  these  organisms  by  the  above  method.. 


252  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

3.  Kleb's  Fractional  or  Dilution  Method.— This  is  a  modification  of  the  ordinary 
plating  methods  introduced  for  use  in  case  the  original  material  is  believed  to  contain 
a  large  number  of  organisms,  and  where  it  is  feared  the  degree  of  bacterial  presence 
is  so  great  that  a  plate  made  from  the  medium  inoculated  with  the  undiluted  substance 
will  be  hopelessly  crowded  with  colonies.  It  is  essentially  the  same  method  of  frac- 
tional or  dilution  inoculation  described  in  connection  with  the  instruction  for  the 
counting  of  bacteria  in  any  material  to  be  analyzed  (page  162).  It  is  very 
popular,  being  perhaps  more  than  any  of  the  other  mechanical  methods  employed 
in  isolation  work.  In  this  practice  it  is  hoped  by  serial  dilutions  of  the  original 
material  to  diminish  the  number  and  thoroughly  disseminate  the  different  types 
of  bacteria  present  so  as  to  obtain  in  a  final  inoculation  growth  of  but  a  single 
variety  or  at  best  of  but  a  few  types.  These  dilutions  of  the  infected  material  may 
be  made  either  in  sterile  water  before  inoculation,  or  by  diffusions  or  smears  in  the 
medium  of  growth  in  the  course  of  inoculation.  The  latter  is  the  method  ordinarily 
employed.  For  example,  in  the  isolation  of  the  mycobacterium  of  diphtheria  from 
the  material  obtained  from  the  throat  of  a  patient,  a  tube  of  liquefied  blood-serum 
is  first  inoculated  by  rubbing  the  swab  on  which  the  exudate  has  been  collected  upon 
the  surface  of  the  medium.  The  swab  is  now  returned  to  its  protective  tube  and  the 
platinum  loop  taken  up.  This,  having  been  sterilized  in  the  flame  and  cooled,  is 
drawn  over  the  surface  of  the  serum  in  this  first  tube  and  then  carried  over  the  surface 
of  sterile  serum  in  a  second  tube.  It  is  again  flamed  and  cooled  and  drawn  over  the 
inoculated  serum  of  the  second  tube,  and  the  infection  adhering  to  it  carried  to  sterile 
serum  in  a  third  tube.  It  is  to  be  supposed  that  in  these  transferences  but  a  small 
number  of  the  original  organisms  have  found  their  way  into  the  third  tube,  and  these 
few  are  probably  widely  scattered  over  the  surface  of  the  serum.  Each  tube  is  marked 
in  proper  manner  and  placed  in  the  incubator.  The  first  growths  to  appear  in  the 
medium,  usually  within  eighteen  or  twenty  hours,  are  the  small  white,  punctate  colonies 
of  Mycobacterium  diphtheria.  They  are  most  definite  and  easily  recognized  in  the 
third  tube ;  and  from  this,  as  soon  as  discovered,  should  be  transferred  with  the  needle 
to  a  fresh  tube  of  serum,  a  stroke  inoculation  being  made.  (A  film  is  also  to  be  prepared 
and  stained  with  Loeffler's  blue  for  confirmation.)  Later,  in  all  three  tubes,  espe- 
cially in  the  first  and  second,  appear  in  greater  or  less  profusion  the  colonies  of  the 
pus  germs  and  other  organisms,  often  to  utter  confusion. 

The  same  principle  is  followed  where  the  dilution  is  made  by  diffusion  in  liquefied 
gelatine  or  agar.  A  small  quantity  of  the  infected  matter,  as  contaminated  water,  is 
planted  by  diffusion  in  a  tube  of  liquefied  gelatine.  From  this  tube  a  small  quantity 
(a  loopful,  or  a  definitely  measured  amount,  representing  a  certain  proportion  of  the 
original  quantity  of  the  fluid  examined,  if  study  of  the  number  of  bacteria  is  also  to 
be  pursued)  is  transferred  to  a  second  tube  of  liquefied  gelatine  and  diffused ;  from  this 
a  similar  quantity  is  transferred  to  a  third  tube  and  likewise  diffused  in  the  liquefied 
medium  in  it.  All  three  tubes  are  plated  in  one  or  other  manner,  and  the  plated 
cultures  placed  in  proper  surroundings  for  development.  In  the  last,  from  the  di- 
minished numbers  of  germs  present,  the  colonies  will  probably  be  well  separated  from 
each  other,  and  transfer  by  means  of  the  needle  to  fresh  medium  possible.  The  first 
and  perhaps  the  second  cultures  will  probably  be  too  crowded  with  growth  to  permit 
advantageous  manipulation,  but  they  will  at  least  serve  as  controls  to  establish  whether 
in  the  third  culture  all  the  types  of  bacteria  in  the  original  material  have  been 
obtained. 


254  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

Exercise  73. — Let  each  student  make  fractional  smears  from  material 
obtained  from  the  fauces  of  a  diphtheritic  patient ;  or  if  this  be  not  at  hand, 
let  the  same  be  done  with  a  mixture  of  known  diphtheritic  organisms  and 
some  of  the  pus  cocci  diffused  in  a  tube  of  bouillon. 

Exercise  74. — Let  each  student  isolate  the  organisms  in  a  loopful  of 
normal  saliva  by  diffusion  in  liquefied  gelatine  and  agar. 

(B)    PHYSIOLOGIC    METHODS. 

In  this  group  of  procedures  advantage  is  taken  of  some  peculiarity  in  the  life- 
history  of  an  organism  sought  to  be  obtained  in  isolation,  either  its  peculiar  resistance 
to  some  influence  capable  of  destroying  associated  bacteria  before  inoculation,  or  some 
method  of  culture  capable  of  restraining  the  development  of  organisms  present  with 
it  in  the  inoculated  medium.  These  physiologic  methods  are  selective,  and  for  the 
most  part  are  employed  where  some  definite  organism  is  sought,  and  are  not  applicable 
for  the  isolation  of  every  species  of  any  complex  mixture. 

i.  Selection  by  'Living  Animal  Tissues. — Advantage  may  be  taken  of  the  fact 
that  the  living  tissues  of  different  animals  by  nature  afford  to  some  organisms  a  favor- 
able opportunity  for  growth,  while  to  others  they  are  resistive  in  variable  degree, 
perhaps  to  the  extent  of  actual  destruction  of  the  germs.  It  is  to  this  fact  largely 
that  we  refer  the  distribution  of  disease  in  different  varieties  of  animals.  If  a  complex 
mixture  of  bacteria  in  which  there  exists  some  form  especially  pathogenic  for  a  certain 
kind  of  animal  be  inoculated  into  an  individual  of  this  species,  it  is  to  be  presumed 
that  the  pathogenic  germs  will,  from  the  favor  of  the  conditions  afforded,  undergo 
rapid  development,  probably  showing  a  specific  selection  for  some  particular  structure 
of  the  animal  body;  while  it  is  probable  that  the  other  organisms,  finding  less  favorable 
surroundings,  will  either  not  develop  at  all  or  but  poorly  (and  in  the  latter  case  be 
restricted  to  the  immediate  vicinity  of  the  point  of  inoculation).  It  then  is  possible,  by 
planting  upon  proper  laboratory  media  from  the  parts  of  the  animal  which  have  been 
especially  invaded  by  the  pathogenic  organisms,  to  obtain  them  free  from  their  former 
associates.  If,  for  example,  one  will  inoculate  beneath  the  skin  of  a  male  guinea-pig 
some  of  the  nasal  discharge  from  a  horse  affected  by  glanders,  containing  a  mixture 
of  Bacterium  mallei  with  various  pathogenic  and  perhaps  other  organisms,  the  former 
rapidly  invade  the  animal,  selecting  the  testicles  as  special  points  of  growth,  and 
shortly  kill  it;  while  the  pus  germs  are  apt  to  remain  restricted  to  the  subcutaneous 
tissues  about  the  point  of  injection.  By  planting  some  of  the  material  obtained  from 
the  more  or  less  necrotic  testicles  upon  the  laboratory  media  a  pure  culture  of  the 
glanders  organism  may  without  special  difficulty  be  obtained. 

Exercise  75. — Early  in  the  work  of  the  class  some  sputum  from  a  con- 
sumptive case  should  have  been  dissolved  in  physiologic  salt  solution  and 
injected  with  a  hypodermic  syringe  into  the  subcutaneous  tissues  of  the 
abdomen  or  chest  of  a  number  of  guinea-pigs.  In  some  of  the  animals  it  is 
probable  that  the  suppurative  changes  caused  by  the  pyogenic  germs  in  the 
sputum  may  destroy  life  at  a  comparatively  early  period,  but  it  may  be 
expected  that  in  other  instances  the  animals  will  in  the  course  of  some  days 
have  overcome  any  general  or  local  influences  of  these  bacteria.  In  such, 


256  LABORATORY  EXERCISES  L\  BACTERIOLOGY. 

at  a  later  period,  usually  about  three  or  four  weeks,  the  evidences  of  tuber- 
cular disease  will  be  manifested  by  the  appearances  of  enlarged  and  case- 
ated  lymphatic  glands  and  by  emaciation  of  the  animals.  Such  an  animal 
is  killed  and  scrapings  planted  with  the  usual  precautions  upon  solidified 
blood-serum  (select  fresh,  undried  medium),  the  substance  being  well 
rubbed  into  the  surface  of  the  serum,  and  the  inoculated  tubes  placed  at 
body  temperature  in  the  incubator.  In  ten  or  twelve  days  pure  growths 
of  Mycobacterium  tuberculosis  should  be  obtained,  and  may  be  identified  by 
'their  gross  and  minute  characteristics  as  set  forth  in  any  of  the  various 
descriptive  works  on  bacteriology. 

2.  Selection  by  Culture  Medium. — In  a  like  manner  the  different  nutrient 
media,  because  of  peculiarity  of  their  organic  matter  or  the  presence  of  restrictive 
or  favoring  inorganic  constituents,  may  exhibit  a  special  fitness  for  the  nourishment 
of  some  particular  germ,  to  the  more  or  less  absolute  exclusion  of  associated  microbes 
and  thus  lead  to  the  early  and  preponderating  development  of  the  former,  while  the 
latter  are  inhibited  from  growth  in  the  culture  either  entirely  or  in  part.  This  may 
be  noticed  in  the  rapid  development  of  the  diphtheritic  organism  on  blood-serum, 
Loeffler's  medium,  or  ascitic  agar  (agar  made  up  with  ascitic  fluid  instead  of  bouillon, 
and  with  five  per  cent,  of  glycerine  added),  the  colonies  of  this  microorganism  appearing 
a  number  of  hours  earlier  on  such  medium  than  those  of  the  commonly  associated  pus 
bacteria;  while  on  ordinary  agar  or  gelatine  this  is  almost  or  quite  reversed. 

For  this  reason  successful  search  for  some  definite  form  of  bacterium  demands 
thoughtful  selection  of  the  media  to  be  employed  for  its  culture.  It  may  not  be,  however, 
that  the  material  selected  will  favor  isolation  by  increase  of  the  rate  of  development  of 
the  organism  sought  for,  or  by  exclusion  of  growth  of  its  associates ;  all  may  grow  with 
equal  rate,  but  the  medium  may  have  caused  the  desired  germ  to  take  on  some  well- 
marked  gross  cultural  peculiarities,  so  that  its  colonies  are  easily  recognized  from  the 
rest  and  their  selection  for  transfer  to  fresh  medium  made  certain. 

In  this  connection  may  be  mentioned  the  practice  of  adding  special  substances  to 
the  nutrient  medium  for  the  purpose  of  inhibiting  the  growth  of  undesired  bacteria 
in  the  culture,  the  substances  thus  used  having  little  or  no  influence  upon  the  develop- 
ment of  the  particular  microorganism  which  it  is  desired  to  obtain  in  pure  culture. 
Carbolic  acid,  hydrochloric  acid,  iodide  of  potassium,  and  other  substances  are  some- 
times added,  with  this  in  view,  to  the  nutrient  upon  which  fecal  matter  is  implanted 
for  the  purpose  of  restraining  the  growth  of  organisms  other  than  Bacillus  coli  or 
Bacillus  typhosus;  the  employment  of  such  substances  also  bringing  out  differences 
in  the  rate  of  development  and  of  gross  cultural  appearance  as  will  permit  the  recogni- 
tion of  these  two  organisms  from  each  other  in  the  culture,  and  consequently  their 
mechanical  separation. 

Exercise  76. — With  the  sterile  needle  make  stroke  or  smear  inoculation 
of  normal  human  feces  upon  a  dish  of  Eisner's  medium  and  grow  for  twenty- 
four  hours  (if  the  medium  be  made  up  with  agar  instead  of  gelatine,  one 
may  place  the  inoculated  dish  in  the  incubator) .  At  the  end  of  this  time  dis- 
tinct colonies  of  a  pale  brownish  tint  may  be  recognized  as  the  growth  of 


258  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

Bacillus  coli,  the  other  bacteria  of  the  fecal  matter  having  probably  entirely 
failed  of  development.  Plant  from  such  colonies  to  other  sterile  media 
for  comparison  with  the  known  characteristics  of  Bacillus  coli. 

Exercise  77. — From  a  known  mixture  of  typhoid  and  colon  bacilli  plant 
in  the  same  manner  upon  the  same  medium.  Note  at  the  close  of  twenty- 
four  hours  the  appearance  of  the  colonies  of  the  colon  bacillus.  After  a 
second  day  the  colonies  of  Bacillus  typhosus  appear  like  tiny,  clear,  color- 
less droplets.  Separate  to  fresh  media  (agar,  gelatine,  and  potato)  and 
study  for  each  organism  the  macroscopic  cultural  features,  and  the  flagel- 
lation, staining  by  Gram's  method,  liquefaction  of  gelatine,  gas  formation  in 
sugar-containing  bouillon,  acid  production,  milk  coagulation,  indol  pro- 
duction, and  the  agglutination  phenomenon  on  addition  of  serum  from  a 
suitable  typhoid  subject. 

3.  Selection  by  Culture  Temperature. — From  reflection  upon  the  differences 
of  optimum  temperature  for  growth  of  the  different  groups  it  may  be  readily  appre- 
ciated that  this  feature  may  be  utilized  in  efforts  to  separate  from  complex  mixtures 
the    psychrophilic,  mesophilic,  or  thermophilic  bacteria  (vide  exercise  35).     It  need 
scarcely  be  added  that,  having  provided  the  condition  of  temperature  requisite  for 
growth  of  some  bacterium  which  it  is  desired  to  separate  from  such  a  mixture,  the 
first  colonies  appearing  in  the  inoculated  medium  are,  other  things  being  equal,  those 
of  the  sought-for  organism.     These  are  at  once  to  be  transferred  to  fresh  medium 
for  inquiry  as  to  perfect  isolation  and  confirmation  of  type,  lest  if  allowed  a  longer 
period  of  development  other  germs  may  follow  if  the  temperature  to  which  the  culture 
has  been  subjected  be  within  the  vital  range  of  such  forms. 

4.  Selection  by  Culture  Atmosphere. — Similarly,  the  possibility  of  growth  in 
the  ordinary  air  or  in  anaerobic  surroundings  may  be  utilized  in  the  separation  of  the 
obligate  forms  of  the  two  varieties  of  bacteria,  it  being  a  matter  of  no  great  difficulty 
to  isolate  and  obtain  in  pure  culture  one  or  other  variety  where  only  the  obligate 
forms  are  present  in  a  mixture.     The  facultative  forms  may,  however,  grow  along 
with  the  aerobes  in  ordinary  air  or  along  with  the  obligate  anaerobes  in  atmospheres  from 
which  oxygen  has  been  displaced  by  hydrogen  or  nitrogen.     In  such  cases  it  may  be  that 
a  difference  in  the  rate  of  development  will  serve  to  render  possible  the  distinction  of 
obligates  from  the  facultative  varieties,  or  a  proper  selection  of  temperature  or  medium 
of  growth  in  connection  with  atmosphere  may  lead  to  the  same  results.     The  capillary 
tubes  of  Salomonsen,  or  Koch's  suggestion  that  on  the  surface  of  a  plated  medium 
in  ordinary  air  a  small  sterile  plate  of  glass  or  mica  be  applied  to  render  the  under- 
lying medium  relatively  anaerobic,  may  be   utilized  in  this  relation ;  but]}  commonly 
the  anaerobic  jar  with  special  interior  atmosphere  or  the  special  anaerobic  tubes  de- 
scribed in  a  previous  lesson  are  employed  in  such  work. 

Exercise  78. — Make  an  infusion  of  ordinary  garden  earth  in  sterile  water 
and  from  this  make  smear  inoculations  on  agar  surfaces.  Grow  in  an  at- 
mosphere of  hydrogen  at  body  temperature.  Observe  after  twenty -four 
hours  the  appearance  of  small,  whitish,  round  colonies  with  delicate  veil- 
like  margins.  Transfer  by  stab  inoculation  to  gelatine  and  agar  in  at- 


260  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

mospheres  of  hydrogen.  After  identification  of  the  gross  characteristics 
as  those  of  Bacillus  tetani,  study  the  minute  appearances,  sporulation,  flag- 
ellation, staining  by  Gram's  method,  coagulation  of  milk,  acid  production, 
gas  production,  and  indol  production  for  comparison  with  the  known  prop- 
erties of  this  organism.  (Note. — It  is  by  no  means  necessary  that  Bacillus 
tetani  be  the  only  anaerobe  likely  to  be  obtained  from  such  source ;  it  is, 
however,  a  common  one,  and  others  which  may  be  obtained  in  mixture  or 
alone  are  to  be  carefully  differentiated  from  it.) 

5.  Cohn's  Heating  Method. — This  procedure  is  intended  for  the  separation 
of  'spore-forming  bacteria  from  vegetative  bacteria,  and  depends  upon  the  well-known 
resistance  of  spores  to  temperatures  fatal  to  ordinary  germs.  An  infusion  known  to 
contain  spores  of  some  organism,  as  well  as  various  vegetative  varieties,  is  boiled  for 
one  minute  (or,  when  it  is  known  that  the  spores  are  very  resistive,  for  a  longer  time), 
by  which  means  all  the  adult  types  of  bacteria  are  usually  destroyed,  most,  if  not  all, 
of  the  spores  being  uninjured  by  the  heat.  It  is  now  quickly  cooled  and  from  it  inocu- 
lations are  made  in  the  usual  manner  in  the  usual  media  and  the  cultures  placed  in 
proper  surroundings  for  development  of  the  spores  (media  best  suited  for  germination 
of  the  spores  are  those  containing  plenty  of  moisture,  as  bouillon).  There  may,  of 
course,  be  spores  of  several  bacterial  varieties  present  in  the  material,  and  in  such 
case  the  colonies  resulting  from  development  of  the  spores  are  to  be  separated  from 
each  other  by  other  suitable  methods. 

Exercise  79. — Make  an  infusion  of  hay  by  placing  a  bunch  of  hay  in  a  jar 
of  water  and  allowing  it  to  soak  over  night  at  incubator  temperature.  The 
following  morning  the  reddish  infusion  is  strained  from  the  hay  through  a 
piece  of  cheese-cloth  and  boiled  for  one  minute.  Cool  by  placing  the  con- 
tainer in  a  vessel  of  cool  water.  Plant  from  the  boiled  fluid  to  a  number  of 
tubes  of  agar,  gelatine,  and  bouillon,  and  allow  to  grow  in  the  ordinary 
atmosphere  at  room  temperature.  In  a  day  or  two  the  characteristic  ap- 
pearances of  the  hay  bacillus  will  appear  without  contamination  with  the 
other  organisms  which  were  probably  present  upon  the  hay. 

Or,  having  strained  the  infusion  from  the  hay,  dilute  it  with  water  until 
it  is  about  1006  in  specific  gravity  and  neutralize  by  the  addition  of  sodium 
hydrate  (litmus  used  as  indicator).  Distribute  the  fluid  in  a  number  of 
sterile  tubes,  boil  for  one  minute,  and  then  place  in  the  incubator  or  in 
room  temperature.  In  a  day  or  two  a  dense  white  surface  growth  of  Bacil- 
lus subtilis  will  have  been  obtained. 


LESSON    IX. 

CLASSIFICATION    AND    IDENTIFICATION    OF    BAC- 
TERIA. 

No  thoroughly  satisfactory  classification  of  the  bacteria  has  as  yet  been  proposed 
and  will  probably  be  wanting  until  our  knowledge  of  their  characteristics  is  sufficiently 
developed  to  render  certain  their  true  generic  relations.  The  older  classifications, 
dealing  with  the  so-called  oligomorphous  varieties,  were  based  entirely  upon  the  shape 
of  the  individual  bacterial  cells,  a  system  continued  to  the  present  by  a  number  of 
writers;  prominent  functional  characteristics,  as  the  power  to  liquefy  gelatine,  serving 
in  a  measure  to  subdivide  the  major  groups.  It  is  not  to  be  supposed  that  there  does 
not  exist  sufficient  natural  basis  for  arrangement  of  these  forms  of  life  into  orders, 
families,  genera,  and  species,  as  is  done  in  case  of  the  higher  vegetables  and  animals ; 
but  it  must  be  acknowledged  that  from  the  insufficiency  of  our  methods  of  study  there 
is  not  as  yet  that  amount  of  information  which  will  permit  a  stable  arrangement  such 
as  is  established  in  the  studies  of  higher  botany  and  zoology.  Whatever  classification 
is  adopted  must  for  the  present,  therefore,  be  regarded  as  tentative.  Probably  the 
best  classification  for  working  purposes  thus  far  suggested  is  that  proposed  by  Migula 
(System  der  Bakterien,  1900) ;  and  this,  with  certain  emendations  by  Chester  (Deter- 
minative Bacteriology,  1901),  and  further  minor  modifications  approaching  more  closely 
to  the  arrangement  of  Migula,  is  adopted  in  these  pages.  It  is  unfortunate  that  its 
generic  system  is  based  upon  a  structural  feature  of  bacteria  wh'ich  is  difficult  of  demon- 
stration and  apparently  not  invariable  throughout  the  life-history  of  the  individual 
cells  or  under  all  conditions — viz.,  the  presence,  number,  and  position  of  flagella.  It 
is  possible,  however,  that  in  the  future  much  of  this  objection  may  be  relieved  by  the 
discovery  of  more  reliable  methods  of  demonstrating  these  fiagellar  appendages  than 
those  now  known,  when  perhaps  it  will  be  found  that  these  organs  are  not  so  variable 
as  would  to-day  appear.  Or  it  is  possible  that  with  the  advance  of  our  knowledge 
of  the  chemistry  of  bacterial  constitution  some  more  constant  feature  may  be  found 
upon  which  to  base  a  classification  of  convenient  genera.  Until  the  acquirement 
of  some  such  desiderata,  however,  the  writer  is  disposed  to  regard  such  an  arrangement 
as  that  of  Migula  with  much  appreciation,  as  of  no  little  use  in  reducing  to  system 
a  study  which  has  been  marked  by  much  confusion  of  excellent  as  well  as  careless 
work. 

Recognizing  the  entire  group  of  the  schizomycetes  or  fission  fungi  as  an  order, 
the  classification  adopted  would  divide  it  into  two  suborders,  the  Eubacteria  (corre- 
sponding roughly  with  the  oligomorphous  bacteria  of  various  writers),  the  individual 
cells  of  the  members  of  which  have  no  granules  of  sulphur  or  bacteriopurpurin  in  their 
constitution;  and  the  Thiobacteria  (roughly,  the  pleomorphous  bacteria  of  writers),  in 
the  cells  of  which  are  to  be  seen  red  or  violet  granules  of  sulphur  or  bacteriopurpurin. 

The  eubacteria  or  true  bacteria  are  divided  into  five  families  from  morphologic 
peculiarity  of  the  cells:  (I)  Coccacece,  spherical  forms;  (II)  Bacteriacea,  straight,  rod- 

262 


264  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

shaped,  unbranched  forms  without  definite  envelope ;  (III)  Spirillacece,  curved  or  spiral 
rods,  unbranched,  without  envelope;  (IV)  ^Iycobacteriace<s,  rod  forms,  often  with 
irregularly  clubbed  ends,  sometimes  forming  filaments,  showing  some  individuals 
with  true  branching,  usually  straight,  but  sometimes  slightly  curved ;  and  (V)  Chlamido- 
bacteriacea,  filamentous  bacteria  composed  of  rod-shaped  cells  and  surrounded  by  a 
distinct  sheath,  without  granules  in  the  cell  contents. 

The  thiobacteria  are  divided  by  Migula  into  two  families:  (I)  Beggiatoacea,  fila- 
mentous bacteria  containing  sulphur  granules  in  the  cell  contents;  and  (II)  Rhodo- 
bacteriacefe,  non-filamentous,  with  bacteriopurpurin  or  sulphur  granules,  red  or  violet 
in  color,  in  the  cell  contents.  The  latter  family  is  further  divided  into  five  subfamilies : 
(A)  Thiocapsacece,  cell  division  in  three  planes;  (B)  Lamprocystacea,  cell  division  first 
in  three,  then  in  two  planes;  (C)  Thiopedacece,  cell  division  in  two  planes;  (D)  Amebo- 
bacteriacece,  cell  division  in  one  plane;  (E)  Chromateaceos. 

The  following  synopsis  of  the  order  indicates  the  relations  of  the  suborders,  families, 
and  genera: 

Order:    SCHIZOMYCETES. 

(A)  Suborder:  EUBACTERIACE^  (without  colored  granules  in  cell  contents;  un- 
colored  except  in  a  very  few  species,  then  faintly,  generally  green). 

I.  Family:  Coccaceae  (globular,  becoming  slightly  elongated  before  cell  division; 

cell  division  in  one,  two,  or  three  directions). 

(a)  Genus:  Streptococcus   (cell   division  in   one   direction,    united  in  chains, 
non -flagellated) . 

(b)  Genus:  Micrococcus  (cell  division  in  one,  two,  or  three  directions  with 
separation  of  cells;  non-flagellated). 

(c)  Genus:  Sarcina   (cell  division  in  three  directions,  united  in  packets  of 
eight;  non-flagellated). 

(d)  Genus:  Planococcus  (cell  division  in  one,  two,  or  three  directions,  cells 
separate;  flagellated). 

(e)  Genus:   Piano sarcina   (cell   division  in   three   directions,  cells   united  in 

packets  of  eight;  flagellated). 

II.  Family:  Bacteriaceae  (cells  straight,  cylindric,  short,  oval  to  rods  and  fila- 

ments; without  sheath;  no  true  branching;  with  or  without  flagella). 

(a)  Genus:  Bacterium  (cells  straight,   cylindric,   oval  to  rods  or  filaments; 
non-motile,  without  flagella;  endospores  present  or  absent). 

(b)  Genus:  Bacillus  (cells  straight,  cylindric,  oval  to  rods  or  filaments;  motile, 

with   flagella   varying  in   number,    peritrichous ;  endospores   present   or 
absent). 

(c)  Genus:  Pseudomonas  (cells  straight,  cylindric,  occasionally  in  short  fila- 

ments;   motile,    flagella    monotrichous    or    amphitrichous;    endospores 
known  in  only  a  few  species). 

III.  Family:  Spirillaceae  (cells  more  or  less  curved;  cell  division  transverse  to 

long  axis  of  cells;  usually  without  endospores;    with  or  without    flag- 
ella; flagella  few,  monotrichous  or  amphitrichous). 

(a)  Genus:  Spirosoma  (cells  rigid,  without  flagella). 

(b)  Genus:  Microspira  (cells  rigid;  one,  rarely  two  or  three  polar  flagella). 

(c)  Genus:  Spirillum  (cells  rigid;  a  bundle  of  polar  flagella). 

(d)  Genus:  Spirochceta  (cells  flexile,  sinuous). 


18 


266  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

IV.  Family:  Mycobacteriaceae  (cells  straight,  short  or  long,  cylindric,  clavate, 

cuneate  in  form ;  at  times  showing  a  true  branching,  or  as  long,  branched 
mycelial  filaments;  no  sheath;  without  endospores,  but  with  formation 
of  gonidia-like  bodies  due  to  transverse  segmentation  of  cells). 

(a)  Genus:  Mycobacterium  (cells  commonly  short,  cylindric  rods,  sometimes 

bent  and  irregularly  swollen,  clavate  or  cuneate;  may  show  Y-shaped 
forms  or  longer  filaments  with  true  branchings;  may  produce  short 
coccoid  elements  which  are  perhaps  gonidia). 

(b)  Genus:  Streptothrix  (cells  commonly  long-branched   filaments;  produce 
gonidia-like  bodies;  form  aerial  hyphae  in  cultures,  causing  resemblance 
to  moulds). 

V.  Family:    Chlamidobacteriaceae    (filaments    composed    of    rod-shaped    cells, 

and  surrounded  by  a  distinct  sheath ;  cell  division  transverse  or  in  three 
directions,  resulting  in  formation  of  gonidia-like  bodies  which  may  or 
may  not  be  motile). 

(a)  Genus:  Leptothrix  (filaments  unbranched;  division  transverse). 

(b)  Genus:  Phragmidiothrix  (filaments  unbranched;  divisions  in  three  direc- 
tions; sheath  scarcely  visible). 

(c)  Genus:  Crenothrix  (filaments  unbranched;  division  in  three  directions; 
sheath  distinct). 

(d)  Genus:  Cladothrix  (filaments  show  false  branching). 


(B)   Suborder:  THIOBACTERIACE^  (cells  show  presence  of  colored  granules,  or 
sometimes  diffuse  coloring,  red  or  violet). 

I.  Family:  Beggiatoaceae  (filamentous;  with  or  without  sheath;  motile  or  non- 

motile  ;  sulphur  granules  in  cell  contents ;  gonidia  formation  not  known) . 

(a)  Genus:  Beggiatoa  (filaments  motile  by  means  of  undulating  membrane; 

segmentation  not  apparent  except  when  stained  with  iodine;  colorless 
or  faintly  reddish-violet). 

(b)  Genus:  Thiothrix  (filaments  non-motile;  surrounded  by  delicate  sheath; 

sulphur  granules  in  cell  contents;  at  ends  of  filaments  rod-shaped  gonidia  ; 
filaments  unequal  in  diameter). 

II.  Family:  Rhodobacteriaceae   (cells  irregular,   globular,   oval,   cylindric,   non- 

filamentous  ;  contents  show  the  presence  of  sulphur  granules  or  bacterio- 
purpurin,  red  or  violet). 

(A)  Subfamily:  THIOCAPSACE^  (cells  divide  in  three  planes). 

(a)  Genus:  Thiocystis. 

(b)  Genus:  Thiocapsa. 

(c)  Genus:  Thiosarcina. 

(B)  Subfamily:  LAMPROCYSTACE;^  (cells  divide  in  three  planes,  then  in  two). 

(a)  Genus:  Lamprocystis. 

(C)  Subfamily:  THIOPEDACE^E  (cells  divide  in  two  planes). 

(a)  Genus:  Thiopedia. 

(D)  Subfamily:  AMEBOBACTERIACE^  (cells  divide  in  one   plane). 

(a)  Genus:  Amebabacter. 

(b)  Genus:  Thiothece. 

(c)  Genus:  Thiodictyon. 

(d)  Genus:  T  hi  o  poly  coccus. 


268  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

(E)  Subfamily:  CHROMATIACE^. 

(a)  Genus:  Chromatium. 

(b)  Genus:  Rhabdochromatium. 

(c)  Genus:  Thiospirillum. 

In  the  study  of  any  unknown  bacterium  the  student  should  have  in  his  possession 
all  the  data  which  are  obtained  in  a  systematic  completion  of  some  blank  form  as 
provided  in  the  Appendix  of  this  volume.  Furnished  with  this  information,  the 
identification  may  be  attempted  by  reference  to  the  analytic  keys  in  various  systematic 
works  on  bacteriology  (Chester,  Migula,  Neumann  and  Lehmann,  Sternberg,  Fliigge, 
Hueppe,  Fischer,  Mace,  et  a/.).  In  case  of  typical  forms  the  recognition  by  such  refer- 
ence is  not  a  matter  of  great  difficulty;  but,  unfortunately,  owing  both  to  the  insuffi- 
ciency of  our  knowledge  arising  from  inefficiency  of  methods  of  demonstration,  faults 
of  description  of  many  species,  and  consequent  confusion  of  system,  and  to  the  greater 
or  less  variability  apt  to  be  met  in  a  given  species,  difficulties  do  arise  for  the  experi- 
enced as  well  as  the  inexperienced.  Patience  and  care  of  observation  are  absolute 
essentials.  As  far  as  they  go,  our  methods,  if  properly  applied,  are  reliable,  and  when 
in  difficulty  the  student  should  recall  the  possibility  of  bacterial  variation  as  giving 
rise  to  his  trouble  and  extend  his  observations  to  other  examples  of  the  species  under 
investigation,  in  hope  of  detecting  the  source  of  apparent  discrepancy.  Methodical, 
painstaking  effort  will  always  yield  some  measure  of  success ;  and  if  certain  recognition 
of  the  species  is  impossible  to  the  student,  at  least  it  will  be  possible  to  refer  temporarily 
and  tentatively  the  bacterium  studied  to  some  class  typified  by  some  well-known 
organism,  after  which  the  subsequent  work  is  narrowed  to  differentiation  from  the 
other  members  of  the  same  class. 

The  scope  of  this  work  scarcely  permits  the  publication  of  any  extended  analytic 
key,  for  which  reference  to  the  descriptive  texts  must  be  made;  however,  for  con- 
venience in  the  preliminary  classification,  the  following  outline,  based  upon  the  above 
table  of  classification,  and  adapted  with  some  changes  from  Chester's  Determinative 
Bacteriology,  is  presented,  leading  to  the  separation  into  genera  of  the  true  bacteria. 
Thereafter  follows  a  list  of  equivalents  of  the  generic  names  to  which  reference  may 
.be  made  in  the  use  of  reference  works  other  than  Chester  in  the  determination  of 
species. 

(A)  Cell  contents  devoid  of  bacteriopurpurin  or 
sulphur  granules,  red  or  violet.  (Chester 
recognizes  among  this  group  a  few  forms  as 
thiospirillum,  thiothrix,  and  a  class  of  pseudo- 
monads  which  contain  sulphur  granules,  which 
in  the  above  table  of  classification  are,  however, 

placed  among  the  thiobacteriacese), EUBACTERIACE^. 

I.  Cells    globular,    free    or    united,    usually    non-     . 

motile,  mostly  without  flagella, Family:  Coccaceae. 

(a)  Cells  without  flagella;  division  in  one  plane 

(cells  united  in  one  direction) , Genus :  Streptococcus. 

(b)  Cells  without  flagella;  division  in  one,  two, 
or  three  planes  to  formation  of  individuals, 
merismopedia  or  zooglea  masses, Genus:  Micrococcus. 


270  LABORATORY  EXERCISES  IN\  BACTERIOLOGY. 

flfci 

(c)  Cells  without  flagella ;  division  in  three  planes 

to  form  packets  of  eight  or  geometric  mul- 
tiples,   Genus:  Sarci-na. 

(d)  Cells   with   flagella;  division   into   separate 

individuals,    Genus:  Planococcus. 

(e)  Cells    with    flagella;    arranged    in    sarcina 

packets,    Genus:  Planosarcina. 

II.  Cells  short  or  long,  straight,  cylindric ;  separate 

or  in  chains;  without  sheath  surrounding 
chains ;  with  or  without  endospores ;  motile  or 
non-motile;  with  or  without  flagella, Family:  Bacteriaceae. 

(a)  Cells  without  flagella ;  endospores  present  or 

absent,    Genus :  Bacterium. 

(b)  Cells  with  flagella;  peritrichous;  endospores 

present  or  absent, Genus :  Bacillus. 

(c)  Cells  with  flagella,  polar;  endospores  usually 

absent,    Genus:  Pseudomonas. 

III.  Cells   more   or   less   spirally  curved;  division 
transverse  to  long  axis  of  cells ;  usually  without 
endospores;  with  or  without  flagella,  usually 

polar,    Family :  Spirillaceae. 

(a)  Cells  rigid,   comma-shaped   to   spiral;  non- 
motile,  without  flagella ;  without  known  endo- 
spores; cells  single  or  in  zooglea, Genus:  Spirosoma. 

(b)  Cells  rigid,  usually  weakly  curved  comma- 
shaped   or  short  spirals;  each   cell   with   one 
(rarely  two  or  three)    polar  flagellum ;  endo- 
spores unknown, Genus :  Micros pira. 

(c)  Cells  rigid,  comma-shaped  or  spiral,  of  vari- 
able  thickness;  cells  actively  motile   with   a 
bunch  of  four  or  more  flagella  at  one  or  both 

poles ;  a  few  forms  with  endospores, Genus :  Spirillum. 

(d)  Long,  slender  filaments;  cells  flexile;  undu- 
latory  or  snake-like  movement  not  progressive 
or  about  the  long  axis ;  flagella  not  known ; 

endospores  apparently  absent, Genus:  Spiroch&ta. 

IV.  Cells  either    short  or  long,   cylindric-clavate- 
cuneate    in    form,    sometimes    showing    true 
branching,  or  as  long,  branched  mycelial-like 
filaments;    filaments    not    surrounded    by    a 
sheath;  without  endospores,  but  with  forma- 
tion of  gonidia-like  bodies  by  transverse  seg- 
mentation of  rods  or  filaments, Family:  Mycobacteriaceae. 

(a)  Cells  ordinarily  short  cylindric  rods,  often 
bent,  irregularly  swollen,  clavate  or  cuneate, 
sometimes  in  Y-shaped  forms  or  in  longer 
filaments  with  true  branchings;  without 
flagella ;  without  endospores,  but  may  present 
short  coccoid  elements,  perhaps  gonidia, Genus:  Mycobacterium. 


272  LABORATORY  EXERCISES  IN  BACTERIOLOGY, 

(b)  Cells  ordinarily  long-branched  filaments; 
non-motile;  without  endospores,  but  produce 
short  gonidia-like  bodies  by  multiple  seg- 
mentation of  filament ;  cultures  on  solid  media 
raised,  dry,  rough,  crumpled,  often  mould-like 

from  formation  of  aerial  hyphae, Genus :  Streptothrix. 

V.  Filamentous  bacteria,  composed  of  rod-shaped 
cells ;  filaments  surrounded  by  sheath ;  cell 
division  into  motile  or  non-motile  gonidia, ..  Family :  Chlamidobacteriaceae. 

(a)  Filaments  unbranched;  non-motile;  inclosed 
in  delicate  or  thick  sheaths ;  fixed  or  in  slimy 
masses.     Cell  contents  become  segmented  into 
round  or  oval  gonidia  by  transverse  division; 
gonidia  non-motile.     (Separation  of  filaments 
and  presence  of  sheath  shown  by  iodine  stain- 
ing),     Genus :  Leptothrix. 

(b)  Filaments  unbranched;  surrounded  by  very 
delicate   sheath   seen   only  in   old   filaments; 
filaments  consist  at  first  of  rods  in  one  plane, 
which  later  divide  in  three  planes  to  form 
sarcina-like   packets;  later  the  cells  become 

spherical  and  free, Genus :  Phragmidiothrix. 

(c)  Filaments  unbranched,  surrounded  by  defi- 
nite sheath,  fixed,  usually  thinner  at  base  than 
at  free  end ;  cell  division  in  three  directions  to 
formation    of    gonidia     (micro-    and    macro- 
gonidia),  which  may  escape  or  germinate  in 

filament, Genus :  Crenothrix. 

(d)  Filaments    show    false    branching,    due    to 
intercalary  growth  of  a  cell  through  the  sheath 
laterally;    filaments    generally    with    delicate 
sheaths,  often  fixed  and  forming  tufts;  repro- 
duction    by    formation     of    motile    gonidia 
(swarm     spores)     which    bear    a    bundle    of 

flagella  laterally  to  one  pole, Genus :  Cladothrix. 

LIST  OF  MORE  OR  LESS  EQUIVALENT  TERMS  IN  GENERIC  NOMEN- 
CLATURE. 

Streptococcus,  Billroth  (diplococcus;  leuconostoc,  VanTieghem;  ascococcus,  Cienkowski). 
Micrococcus,     Hallier-Cohn     (coccus;     sph&rococcus;     tetrad;     merismopedia;     merista; 

pediococcus;  staphylococcus,  Ogston;  leucocystis,  Schrotter;  hyalococcus,  Schrotter; 

lampropedia,     Schrotter;     bacteridium,     Schrotter;    ascococcus,     Billroth;    monas, 

Ehrenberg) . 
Sarcina,  Goodsir. 

Planococcus,  Migula  (id.  micrococcus). 
Piano sarcina,  Migula. 
Bacterium,    Ehrenberg-Migula   (bacillus,   Cohn ;  bacteridium,    Davaine ;   Neumann  and 

Lehmann  and  others  reserve  the  name  for  rods  devoid  of  endospores). 


274  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

Bacillus,  Cohn-Migula  (bacterium,  Huppe;  Neumann  and  Lehmann  and  others  employ 
the  term  as  indicating  rods  containing  endospores;  includes  special  forms,  as 
clostridium;  paraplectrum;  ascobacillus;  proteus,  Hauser;  halibacieria,  etc.,  Fischer). 

Pseudomonas,  Migula  (terms  applied  to  last  two  genera ;  monas;  includes  the  class 
nitromonas  of  Winogradsky) . 

Spirosoma,  Migula  (comma-bacillus;  spirillum,  Ehrenberg;  microspira,  Schrotter; 
vibrio,  Miiller). 

Microspira,  Migula  (spirillum,  Ehrenberg;  vibrio,  Miiller;  comma-bacillus}. 

Spirillum,  Ehrenberg-Migula  (vibrio,  Miiller;  comma-bacillus). 

Spirocheeta,  Ehrenberg. 

Mycobacterium,  Lehmann  and  Neumann  (includes  corynebacterium,  Lehmann  and 
Neumann;  sclerothrix,  Metschnikoff ) . 

Streptothrix,   Kruse  (actinomyces,   Harz ;  cladothrix,  Giinther ;  odspora,  Wallroth) . 

Leptothrix,  Kiitzing  (streptothrix,  Migula). 

Exercise  So. — Let  the  instructor,  having  placed  upon  the  blackboard 
the  characteristics  of  some  known  microorganism,  require  of  each  student 
the  practice  of  identification  of  the  bacterium  from  the  data  given,  follow- 
ing some  selected  analytic  key;  repeat  with  the  characteristics  of  other 
bacteria  as  desired. 


LESSON  X. 

STUDY  OF  THE    PATHOGENIC  ACTION    OF  BACTERIA. 

While  all  bacteria  are  necessarily  of  general  interest  as  being  either  indirectly 
or  directly  important  to  our  life  and  relations,  those  which  produce  disease  in  man 
are  of  essential  interest  to  medical  men.  -To  these  the  name  pathogenic  bacteria  is 
commonly  applied,  the  remainder  being  termed  non-pathogenic  bacteria.  The  known 
pathogens  form  a  comparatively  small  proportion  of  the  numerous  species  thus 
far  discovered;  but  it  should  not  be  forgotten  that  we  have  little  idea  of  the  possi- 
bility of  pathogenic  power  which  under  even  apparently  unimportant  modifica- 
tion of  condition  may  reside  in  the  so-called  non-pathogens.  Anthrax  is  ordinarily 
non-pathogenic  to  chickens,  whose  body  temperature  is  comparatively  high;  but 
should  the  hen's  body  heat  be  lowered  several  degrees  by  refrigeration,  she  becomes 
susceptible  to  the  influences  of  the  bacterium  of  this  disease.  The  relative  amount 
of  acid  or  alkaline  substances  which  Microspira  comma  meets  in  the  alimentary  canal 
of  man  apparently  determines  whether  he  shall  escape  or  be  stricken  with  Asiatic 
cholera;  and  Bacillus  coli,  a  constant  and  profuse  parasite  of  man's  intestinal  canal, 
is  often  met  in  other  situations  in  the  body,  apparently  the  cause  of  serious  lesions. 
It  is  not  impossible  that  every  bacterium  may  somewhere  in  the  range  of  higher  life, 
animal  or  vegetable,  find  itself  capable  of  a  parasitic  existence;  such  parasitism  may 
doubtless  be  forced  by  transient  and  perhaps  unobserved  special  circumstances  or 
conditions.  Parasitism  does  not  necessarily  imply  pathogenesis ;  but  should  it  happen 
that  the  host  is  susceptible  to  the  various  influences  exerted  by  the  parasite  and  its 
products,  then  disease  must  arise.  It  is  from  such  a  view  not  a  wonderful  thing  that 
diseases  unusual  to  man  should  occasionally  appear,  their  entrance  and  development 
depending  upon  some  accident  or  some  temporary  and  unusual  condition;  nor  can 
it  be  looked  on  as  impossible  that  totally  new  disease  occurrence  may  take  place, 
depending  upon  a  forced  invasion  of  some  microbe  hitherto  unknown  as  a  human 
parasite  and  its  development  favored  by  some  unusual  condition  which  may  exist 
in  the  body  of  the  affected  individual.  Correlatively,  while  we  are  accustomed  to 
speak  of  certain  microbes  as  being  obligate  parasites,  this  is  probably  only  in  a  general 
sense  correct;  in  strict  meaning  it  can  scarcely  be  doubted  that  under  unknown  but 
definite  conditions  the  parasites  of  man  might  be  reduced  to  parasitism  in  some  other 
species  or  even  to  a  saprophytic  existence.  Thus  is  explained  the  fact  that  human 
disease  of  some  sort  may  sometimes  apparently  die  out  and  be  unknown  for  a  greater 
or  less  period,  returning,  when  conditions  favor,  to  the  surprise  and  consternation 
of  men. 

It  is  well,  therefore,  to  think  of  all  bacteria  as  having  a  possible  pathogenic  in- 
fluence sometimes  and  to  some  species  of  created  beings;  and  no  matter  what  the 
source  of  the  microorganism  studied,  a  part  of  that  study  forced  upon  us  for  our  own 
protection  must  concern  itself  with  the  influence  the  bacterium  may  exert  upon  higher 
life.  It  is  impossible,  of  course,  that  its  relations  to  all  forms  of  life  be  investigated  ; 

276 


278  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

as  a  rule  we  are  satisfied  with  experimentation  upon  one  or  two  species  of  the  smaller 
animals  easily  available  for  laboratory  work,  as  frogs,  hens,  pigeons,  mice,  rats,  guinea- 
pigs,  rabbits,  and  dogs.  This  part  of  the  study  is  dictated  in  the  third  of  Koch's 
postulates  relating  to  the  study  of  pathogens  and  the  proof  of  their  causal  relationship 
to  disease:  "The  bacterium  in  pure  culture  is  to  be  inoculated  into  a  susceptible  animal 
and  its  effects  compared  with  and  recognized  as  similar  or  identical  to  the  known  symptoms 
of  the  originally  diseased  individual." 

The  selection  of  animals  susceptible  to  the  influence  of  an  organism  is  by  no 
means  always  easy  or  possible  in  the  small  range  available  to  the  laboratory  worker. 
In  general,  however,  it  may  be  said  that  frogs  are  well  suited  for  the  psychrophilic 
bacteria,  and  the  small  warm-blooded  animals  for  the  mesophilic  varieties.  After 
inoculation  the  animals  are  to  be  kept  in  as  nearly  normal  relations  and  surroundings 
as  possible,  and  it  is  well  always  to  place  in  the  same  conditions,  but  in  separate  con- 
tainers, one  or  more  similar  but  uninoculated  animals  as  controls.  The  inoculations 
may  be  made  in  one  or  other  of  a  variety  of  ways.  Sometimes  a  mere  scratching 
of  the  skin,  as  in  vaccination,  with  the  culture  laid  on  the  surface  and  well  rubbed 
in  with  a  small  spatula,  is  sufficient.  Or  a  bit  of  thread  moistened  with  a  fluid  culture 
may  be  drawn  through  the  skin  after  the  manner  of  applying  a  seton.  Usually,  how- 
ever, more  definite  operations  are  required,  and  are  preferable  from  more  or  less  cer- 
tainly avoiding  the  entrance  of  the  common  bacteria  of  the  skin  as  contaminating 
and  confusing  influences,  hypodermic,  intravenous,  and  intraperitoneal  inoculations 
being  the  usual  forms  in  the  warm-blooded  animals. 

Hypodermic  Inoculations. — Mice  are  best  held  by  the  end  of  the  tail  in  a  glass 
jar,  the  tail  being  drawn  out  over  the  edge  of  the  jar  and  the  mouse  held  in  the  jar 
by  a  board  over  the  top  of  the  latter  so  placed  as  to  permit  the  exposure  of  the  tissues 
at  the  base  of  the  tail  only.  Guinea-pigs  and  rabbits  may  be  held  by  an  assistant, 
the  two  hind  legs  in  one  hand,  the  two  fore  legs  in  the  other;  or  one  of  the  various 
mechanical  animal  holders  (Fig.  68)  may  be  employed.  Dogs  had  best  be  fastened 
for  any  important  inoculation  in  one  of  the  troughs  commonly  used  for  this  purpose 
in  experimental  work,  although  for  inoculation  with  the  syringe  this  is  scarcely  neces- 
sary. Pigeons  and  hens  should  be  held  with  one  hand  grasping  the  legs,  tips  of  the 
wings,  and  the  tail,  and  the  other  holding  the  neck  in  extension.  Mice  and  rats  are 
usually  inoculated  in  the  tissues  about  the  base  of  the  tail  and  along  the  lower  end 
of  the  spine;  rabbits  and  guinea-pigs,  in  the  tissues  of  one  side  of  the  thorax;  pigeons 
and  hens,  in  the  pectoral  region;  the  culture  being  usually  introduced  into  one  of 
the  subcutaneous  lymph-sacs  when  frogs  are  used  as  experiment  animals. 

It  is  next  to  impossible  to  thoroughly  sterilize  the  skin,  but  the  hair  or  fur  should 
be  removed  by  clippers  and  razor  or  the  feathers  plucked  from  a  small  area  about 
the  site  of  the  proposed  inoculation  and  the  surface  well  washed  with  soap  and  water 
and  rinsed  with  boiled  water  (or  a  pledget  of  cotton  soaked  with  a  1 :  1000  solution 
of  corrosive  sublimate  applied  closely  to  the  surface  for  a  few  minutes,  alcohol  and 
ether  being  afterward  used  for  rinsing).  The  culture  may  be  suspended  in  sterile 
water  or  physiologic  salt  solution,  or  an  active  bouillon  culture  employed,  when  it  is 
best  introduced  beneath  the  skin  by  means  of  a  syringe.  Or  a  dry  culture  may  be 
inserted  into  a  small  pocket  under  the  skin  upon  a  stiff  platinum  needle.  The  hypo- 
dermic syringe  used  should  be  so  constructed  that  it  may  be  thoroughly  sterilized, 
made  of  glass,  metal,  and  rubber  (as  Koch's  or  Strohschein's).  An  excellent  substitute 
may  be  extemporized  from  a  glass  pipette  or  Sternberg's  bulb  with  a  hypodermic 
needle  attached  to  the  tube  by  means  of  a  rubber  tube,  the  fluid  being  forced  from 


280 


LABORATORY  EXERCISES  IN  BACTERIOLOGY. 


the  pipette  by  blowing  into  the  upper  end,  which  is  plugged  with  a  sterile  cotton  stopper, 
and  from  the  bulb  by  the  application  of  heat.  The  syringe  and  all  its  parts  should 
have  been  sterilized  by  boiling  or  in  the  autoclave,  or  by  prolonged  immersion  in  a 
disinfecting  solution,  with  subsequent  rinsing  in  sterile  water.  The  fluid  in  wrhich 
the  bacteria  are  suspended  having  been  drawn  into  the  syringe,  a  fold  of  the  skin  is 
pinched  up  with  the  fingers  or  a  pair  of  forceps  and  the  needle  is  thrust  into  the  sub- 
cutaneous tissues  of  the  fold,  penetrating  some  distance  from  the  point  of  entrance. 
The  fluid  is  then  forced  gently  and  steadily  into  the  tissues  and  the  needle  then  with- 
drawn. It  is  usually  unnecessary  to  do  anything  more ;  but  if  desired,  a  drop  of  collodion 
may  be  applied  to  the  point  of  puncture. 


FIG.  68. — DIFFERENT  FORMS  OF  ANIMAL  HOLDERS. 


In  introducing  a  solid  culture  under  the  skin,  after  having  cleansed  the  surface 
as  in  the  previous  description,  it  is  necessary  to  cut  with  a  sterile  scissors  a  small  fold 
of  the  skin  pinched  up  by  the  fingers  and  then  holding  the  edge  of  the  incision  with 
a  sterile  forceps  to  break  a  small  pocket  with  any  convenient  sterile  instrument  in  the 
subcutaneous  tissues,  into  which  the  culture  is  carried  on  a  stout  platinum  needle ; 
the  skin  wound  is  then  closed  with  a  collodion  dressing. 

It  is  impossible,  without  some  previous  knowledge  as  to  the  degree  of  virulence 
of  a  bacterium,  to  determine  precisely  the  dosage  of  the  material  to  be  inoculated  into 
an  animal;  however,  as  a  general  rule  for  subcutaneous  injections  it  is  customary  to 
employ  about  one  per  cent,  of  the  body  weight  of  the  experiment  animal  of  an  active 
bouillon  culture. 

Intravenous  Inoculation. — This  is  generally  performed  on  rabbits,  the  vein 
along  the  posterior  border  of  the  ear  of  this  animal  being  an  easy  one  for  entrance 


19 


282  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

with  a  fine  hypodermic  needle.  The  animal  as  a  rule  requires  little  holding.  The 
needle  is  introduced  from  the  dorsal  side  of  the  ear.  It  is  scarcely  necessary  to  shave 
the  little  hair  from  the  skin,  which  is,  however,  to  be  well  washed  to  begin  with ;  where 
the  hair  is  at  all  marked,  however,  it  is  best  to  shave  the  ear  and  wash  it  thoroughly. 
One  must  usually  compress  the  base  of  the  ear  in  order  to  make  the  vein  in  its  course 
prominent ;  a  small  rubber  band  about  the  root  of  the  ear  serves  this  purpose  well. 
The  fluid  in  the  syringe  is  carefully  brought  to  the  end  of  the  needle  so  that  no  bubbles 
of  air  will  be  carried  in  the  process  into  the  circulation.  The  needle  is  then  introduced 
into  the  vein  as  near  the  tip  of  the  ear  as  practicable  where  the  vein  is  straightest  in 
its  course,  care  being  taken  that  it  really  enters  into  the  interior  of  the  vessel.  The 
fluid  is  then  forced  gently  and  steadily  into  the  vein,  blanching  its  previous  red  line 
as  the  blood  is  displaced.  Before  the  needle  is  withdrawn  the  pressure  at  the  base 
of  the  ear  should  be  released;  and  after  withdrawal  the  tiny  opening  may  be  sealed 
with  collodion.  A  similar  operation  may  be  practised  in  case  of  the  veins  of  a  dog's 
ear;  in  other  animals  and  in  deeper  seated  vessels  it  is  necessary  to  expose  the  vein 
by  definite  dissection. 

Intraperitoneal  Inoculation. — This  may  be  performed  with  the  hypodermic 
syringe;  or  an  opening  may  be  made  by  regular  surgical  operation  and  through  this 
a  solid  material  introduced  into  the  cavity. 

The  first  procedure  is  relatively  simple.  The  animal  is  fastened  upon  one  of  the 
mechanical  holders,  the  hair  or  fur  removed  from  a  small  area  of  the  abdominal  surface 
by  means  of  the  clippers  and  razor,  and  the  surface  well  washed.  The  needle  of  the 
syringe  holding  the  inoculating  material  is  inserted  under  the  skin  as  in  subcutaneous 
injections,  and  then  the  abdominal  wall  is  firmly  pinched  up  into  a  comparatively 
large  fold,  this  practice  removing  the  danger  of  puncturing  the  intestine.  The  needle 
is  next  thrust  well  into  the  fold  until  its  point  is  felt  to  be  free  in  the  cavity,  when  the 
fluid  is  forced  into  the  latter.  The  needle  is  now  withdrawn  and  a  drop  of  collodion 
applied  to  the  skin  puncture  (which  is  not  in  exact  correspondence  with  the  puncture 
into  the  cavity,  the  latter  being  protected  by  the  skin  at  a  little  distance  from  the 
point  of  entrance  into  the  cutaneous  surface). 

In  opening  the  abdomen  for  the  introduction  of  solid  infections  the  animal  is 
similarly  prepared.  The  skin  having  been  freed  from  hair  and  fur  and  the  surface 
cleansed,  the  site  of  operation  is  anesthetized  by  cocaine,  after  which  an  incision  is 
made  through  the  skin  with  a  sterile  blade  or  scissors  and  the  skin  reflected  from  the 
underlying  tissues.  The  incision  is  to  be  made  in  the  median  line  in  order  to  minimize 
the  danger  of  bleeding.  The  tissue  of  the  full  thickness  of  the  exposed  abdominal 
wall  is  now  pinched  up  into  a  fold  by  the  fingers  and  several  sutures  corresponding  to 
the  size  of  the  opening  to  be  made  are  inserted  through  the  entire  thickness,  passing 
in  and  out  of  the  peritoneum  in  their  course ;  these  sutures  are  to  be  made  transversely 
to  the  line  of  incision  in  making  the  opening.  This  done,  a  small  incision  is  cut  with 
a  sterile  instrument  so  as  to  open  the  cavity  and  through  this  the  inoculation  material 
introduced.  The  opening  through  the  wall  is  now  closed  by  adjustment  of  the  sutures; 
after  which  the  skin  is  drawn  into  place  and  closed  by  means  of  a  second  suturing 
and  the  line  of  the  skin  wound  sealed  with  a  collodion  dressing. 

Other  Forms  of  Inoculation. — Inoculations  are  sometimes  made  into  the  anterior 
chamber  of  the  eye,  where  the  development  of  the  consequent  local  lesion  may  be  watched. 
In  this  case  the  lids  are  held  by  proper  retractors  and  the  surface  of  the  conjunctiva 
irrigated  with  a  solution  of  bichloride  of  mercury  and  afterward  with  sterile  water; 
after  which,  with  a  sterile  cataract  needle,  the  cornea  along  the  junction  with  the 


284  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

sclera  is  incised  and  through  the  opening  thus  made  the  infection  introduced  by  syringe 
or  on  a  small  sterilized  forceps. 

Inoculations  into  the  cerebro-spinal  area  are  made  after  performance  of  a  definite 
surgical  opening  of  the  cranium  by  trephine  or  chisel,  the  operation  being  performed 
somewhat  to  one  side  of  the  sagittal  line  in  order  to  avoid  the  longitudinal  sinus. 
Through  this  opening  a  sterile  forceps  grasps  the  dura  mater  and  draws  it  slightly 
upward  in  a  small  fold  as  well  as  may  be  done ;  through  the  fold  or  the  adjacent 
tense  part  of  the  membrane  the  needle  of  the  syringe  is  passed  into  the  arachnoid 
space,  into  which  the  fluid  and  its  contained  bacteria  are  forced  slowly;  the  trephine 
opening  is  covered  by  the  replacement  of  the  reflected  overlying  tissues  and  the 
external  wound  dressed  as  in  ordinary  surgery. 

In  inhalation  inoculations  the  animal  is  placed  in  a  small  closed  chamber,  into 
the  atmosphere  of  which  the  fluid  in  which  the  bacteria  are  suspended  is  vaporized 
mechanically,  and  the  animal  left  in  the  chamber  for  a  time  to  breathe  the  air  con- 
taminated with  the  infection. 

Alimentary  inoculation  is  accomplished  by  mixing  some  of  the  culture  with  the 
food  and  then  allowing  the  animal  to  feed  upon  it.  Occasionally  enemata  containing 
the  infected  matter  are  thrown  into  the  lower  intestinal  tract. 

After  inoculation  has  been  performed  the  experiment  animals  should  receive 
regular  and  frequent  observation  as  to  the  condition  of  the  inoculation  site,  the  presence 
of  other  apparent  structural  alterations,  and  the  presence  of  any  general  or  special 
symptoms.  The  temperature  of  the  animal  should  be  taken  by  rectum  at  regular 
intervals  and  charted,  the  movements,  attitude,  appetite,  excretions,  and  the  general 
appearance  and  habits  of  the  animal  watched  and  recorded;  and  the  occurrence  of 
spasms,  lethargy,  or  other  appreciated  phenomena  should  be  made  a  matter  for  record. 
If  one  be  studying  an  organism  derived  from  a  diseased  individual,  all  these  peculiarities 
are  to  be  carefully  compared  with  the  symptoms  observed  in  the  original  disease  and 
their  similarity  or  identity  established;  but  it  is  to  be  recollected  that  the  difference 
in  the  experiment  animal  from  the  originally  diseased  individual  and  the  mode  of 
artificial  inoculation  may  make  considerable  difference  in  the  clinical  pictures  of  the 
two  cases. 

Disease  production  by  bacteria  may  be  brought  about  through  several  possible 
separate  or  combined  influences.  They  may  (a)  cause  irritation  by  their  presence 
or  their  active  movements  in  the  tissues  in  some  locality  (or  if  numerous  points  of 
irritation  be  induced,  a  more  or  less  general  inflammatory  condition  may  be  present)  ; 
(6)  they  may  act  untowardly  by  the  abstraction  of  important  elements,  as  oxygen, 
from  the  system  (either  the  bacteria  or  their  products) ;  (c)  when  in  great  numbers 
in  some  passageway,  as  a  small  vessel,  they  may  cause  its  obstruction  and  thus  perhaps 
interfere  with  nutrition  or  some  other  important  function ;  and  (d)  they  may  elaborate 
or  cause  the  formation  from  the  body-elements  of  principles  which  are  poisonous  to 
the  host  (vide  Lesson  VII,  section  Alkaloidal  Products).  The  most  important  of 
these  modes  of  operation  is  the  last,  to  which  are  due  principally  the  general  symptoms 
of  disease,  as  well  as  the  various  degenerative  changes  of  the  tissues;  to  the  first  of 
these  influences  are  due  in  large  measure  the  inflammatory  changes  met  about  the 
site  of  inoculation. 

After  the  death  of  the  animal  (which  may  be  killed  upon  definite  development 
of  evidence  of  the  presence  of  disease)  a  careful  autopsy  should  be  made  in  order  to 
establish  the  alterations  which  have  been  produced  in  the  tissues  and  to  render  certain 
that  the  organisms  inoculated  have  been  the  true  or  probable  cause  of  the  disease 


286  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

following  the  inoculation  (this  is  indicated  by  their  discovery  in  isolated  condition 
in  increased  numbers  in  the  body  of  the  animal).  This  last  constitutes  the  fourth 
of  Koch's  postulates  for  the  proof  of  the  causal  relation  of  a  given  germ  to  disease: 
"From  the  fluids  and  tissues  of  the  experimentally  diseased  animal  the  same  organism 
inoculated  must  be  recovered." 

The  autopsy  should  be  conducted  as  soon  after  death  as  possible,  lest  contaminat- 
ing organisms  from  the  intestinal  canal  or  putrefying  germs  find  their  way  into  the 
tissues  and  fluids  of  the  body;  if  it  is  impracticable  to  perform  the  operation  at  once, 
the  dead  animal  should  be  refrigerated  in  order  to  prevent  such  occurrence.  In 
conducting  the  autopsy  the  utmost  care  to  prevent  contamination  must  be  observed. 
The  usual  preliminary  observations  as  to  the  body,  general  appearance,  external 
lesions  and  marks,  body  weight,  degree  of  nutrition,  temperature,  rigidity,  condition 
of  site  of  inoculation,  etc.,  are  first  made.  The  animal  is  then  laid  on  its  back  on  a 
convenient  board  and  the  legs  extended  and  fastened  to  the  board  by  means  of  tacks 
or  staples.  The  skin  is  well  wet  with  a  solution  of  bichloride  of  mercury,  and,  a  median 
incision  having  been  made  from  the  pubic  region  to  the  throat,  the  skin  is  dissected 
well  off  the  abdomen  and  thorax  and  tacked  out  on  the  board  so  as  to  remove  the 
likelihood  of  accession  of  bacteria  from  it  to  the  parts  to  be  exposed.  The  site  of  in- 
jection of  the  infection  and  any  other  subcutaneous  lesions  are  now  inspected,  the 
removal  of  the  skin  affording  opportunity  for  close  observation ;  and  from  the  inocula- 
tion site  are  made  a  number  of  inoculations  on  various  nutrients,  smears  on  glass 
slides  are  prepared  for  microscopic  examination,  and  bits  of  the  tissue  are  placed  in 
absolute  alcohol,  where  they  are  fixed  for  section-making.  The  body  cavities  are  now 
opened  by  the  usual  longitudinal  incision  in  the  median  line,  with  such  additional 
lateral  incisions  as  may  be  required  for  inspection  of  the  interior,  with  a  sterile  pair 
of  scissors  (boiled  and  kept  in  boiled  water).  On  opening  the  cavities,  inoculations 
and  smears  for  microscopic  examination  are  made  from  any  exudate  encountered ; 
and  subsequently  inoculations  are  to  be  made  and  smears  prepared  on  glass  slides 
from  each  of  the  important  organs  and  the  blood  as  detailed  in  Lesson  V.  Likewise, 
small  bits  of. tissue  from  all  suspicious  points  and  the  principal  organs  are  fixed  in 
absolute  alcohol  for  sections.  The  remains  of  the  animal  should  then  be  cremated, 
the  autopsy  board  well  disinfected,  and  the  hands  of  the  operator  disinfected  and 
well  washed  with  soap  and  water.  (In  case  of  severe  infections  it  is  a  wise  practice 
to  have  the  hands  incased  in  rubber  gloves  during  the  above  manipulations.) 

The  subsequent  study  of  the  material  thus  obtained  concerns  the  discovery  of 
the  organisms  in  the  film  preparations  and  in  the  sections  of  the  tissues,  their  isolation 
from  some  or  all  of  the  points  from  which  inoculation  to  nutrient  media  was  made 
(by  culture  of  the  inoculated  media),  and  the  recognition  of  destructive,  degenerative, 
or  inflammatory  changes  in  the  various  structures;  all  of  which  are  to  be  compared 
with  the  alterations  known  to  have  been  produced  by  the  original  disease  in  case 
the  infection  was  derived  from  a  diseased  individual. 

Should  the  experiment  animal  or  animals  have  recovered  from  the  effect  of  the 
inoculation,  after  some  days  it  is  well  to  repeat  the  same  with  a  view  of  recognizing 
any  degree  of  immunity  which  may  have  been  acquired  from  the  original  attack.  We 
are  accustomed  to  speak  of  the  natural  protection  possessed  by  an  individual  against 
the  effects  of  a  given  virus  as  his  natural  immunity  against  that  germ,  contrasting 
with  it  the  protection  gained  by  artificial  means  or  through  a  prior  attack  of  the  same 
disease  (acquired  immunity).  Natural  immunity  against  the  invasion  and  influences 
of  bacteria  in  general  depends  on  one  or  a  number  of  the  following  factors:  (a)  The 


288  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

protection  against  invasion  of  the  germs  afforded  by  the  skin,  mucous  membranes, 
and  other  protective  surfaces ;  (6)  the  action  of  the  cilia  present  on  the  cells  of  certain 
membranes  tending  to  expel  the  organisms  seeking  entrance;  (c)  the  influences  of  the 
various  secretions  with  which  the  bacteria  may  come  in  contact,  as  the  acid  juice 
of  the  stomach  or  the  alkaline  juice  of  the  small  intestine ;  (d)  the  destructive  (phago- 
cytic)  action  of  the  white  blood-cells  and  the  embryonic  connective-tissue  corpuscles; 
(e)  the  influences  of  certain  agents  antagonistic  to  bacterial  development  or  their 
products,  present  naturally  in  the  fluids  of  the  body  (alexins  or  natural  antitoxins'). 
It  is  believed  that  if  these  influences  be  sufficiently  strong  an  invading  organism  is 
destroyed  without  toeing  able  to  manifest  its  presence  by  the  production  of  noticeable 
disease;  and  the  limitation  of  the  ordinary  acute  infections  is  looked  upon  as  being 
due  to  the  development  of  sufficiently  strong  action  of  the  phagocytes,  alexins,  or  anti- 
toxic substances  formed  in  the  system  of  the  diseased  individual  during  the  course 
of  the  infection.  (Possibly,  also,  the  exhaustion  of  some  substances  necessary  for  the 
growth  of  the  infection  may  aid  in  this  limitation  of  the  disease  course.) 

Should  the  protection  thus  brought  about  be  complete,  so  as  to  entirely  prevent 
the  reacquirement  of  the  disease,  such  acquired  immunity  is  said  to  be  absolute;  if, 
however,  it  permit  the  return  of  the  disease,  only  in  a  less  degree  of  severity,  it  is  said 
to  be  partial.  Advantage  is  taken  of  the  possibility  of  acquiring  such  protection  to 
induce  it  artificially  (a)  by  "inoculation"  with  the  natural  virus  of  the  disease  in  ques- 
tion when  known  to  be  of  a  mild  type;  (6)  by  introducing  the  virus  after  it  has  in 
some  way  been  purposely  weakened  and  is  unable  to  induce  the  severe  manifestations 
of  the  disease,  but  is  yet  able  to  confer  the  desired  immunity  (vaccination) ;  or  (c)  by 
the  introduction  into  the  system  of  antitoxic  material  derived  from  individuals  pre- 
viously influenced  by  the  disease  itself  or  by  the  filtered  toxins  of  the  disease  (as  in 
the  use  of  diphtheria  antitoxin  obtained  from  horses  which  have  been  subjected  pre- 
viously to  the  filtered  toxins  of  cultures  of  Mycobacterium  diphtheria  until  they  no 
longer  react  to  large  doses  injected  into  them).  The  immunity  acquired  by  such 
methods  is  spoken  of  as  artificial  immunity;  other  means  are  now  and  again  available, 
as  the  influence  of  drugs,  or  rarely,  the  antagonism  exhibited  by  some  associated 
organism  to  the  development  or  effects  of  the  virus  in  question.  The  immunity  which 
is  acquired  by  an  attack  of  disease  (either  the  severe  natural  affection,  or  the  mild 
natural  disease  transmitted  to  the  individual  in  the  practice  of  "inoculation,"  or 
the  purposely  weakened  disease  induced  in  vaccination)  is  likely  to  be  more  or  less 
persistent,  the  antitoxic  substances  upon  which  it  depends  being  produced  within  the 
system  of  the  individual  and  in  some  instances  continued  for  an  indefinite  period 
after  the  attack ;  such  immunity  is  said  to  be  an  active  immunity.  When,  on  the  other 
hand,  the  protection  is  afforded  by  the  introduction  of  immunizing  substances,  as 
antitoxin,  from  another  individual,  or  of  drugs,  into  the  system,  the  effect  lasts  only 
during  the  persistence  of  the  dose  administered ;  when  the  material  has  been  excreted 
or  destroyed  in  the  system,  the  protection  no  longer  exists.  Such  immunity  is  spoken 
of  as  passive. 

Note. — The  following  exercises,  for  the  sake  of  time,  should  have  been  inaugurated 
one  or  two  weeks  before  the  close  of  the  class  work;  and  provision  for  them  should  be  made 
as  soon  as  the  class  has  come  to  understand  the  conditions  of  bacterial  culture. 

Exercise  Si. — Bach  two  students  working  together  inoculate  intra- 
venously a  rabbit  with  M.  pyogenes  aureus.  In  fatal  cases  let  formal 


290  LABORATORY  EXERCISES  IN  BACTERIOLOGY. 

autopsy  be  conducted,  and  from  the  metastatic  abscesses  let  nutrient 
tubes  be  inoculated  and  films  prepared  for  microscopic  examination.  In 
young  animals  note  the  occurrence  of  osteomyelitis. 

Exercise  82. — Before  the  class  let  the  instructor  inoculate  several 
guinea-pigs  with  a  bouillon  culture  of  Bad.  anthracis  grown  for  three  weeks 
at  a  temperature  of  42°  C.  Let  the  animals  be  exhibited  to  the  class 
during  the  period  of  reaction  and  after  recovery.  After  recovery  let  them 
be  inoculated  with  a  bouillon  culture  of  the  same  organism  grown  at  the 
same  temperature  for  two  weeks;  and  after  recovery  from  this  let  the 
same  operation  be  performed  upon  them  with  a  similar  culture  grown 
for  but  one  week  at  the  same  elevated  temperature.  After  several  days 
from  the  last  inoculation  let  such  an  animal  and  one  which  has  not  been 
subjected  to  the  vaccines  be  inoculated  with  virulent  anthrax  to  demon- 
strate the  acquirement  of  immunity  by  the  influence  of  the  vaccine. 

Exercise  83. — Inject  a  guinea-pig  of  about  two  hundred  and  fifty  grams 
weight  with  one  protective  unit  of  diphtheria  antitoxin  (amount  of  anti- 
toxin requisite  to  counteract  one  hundred  fatal  doses  of  the  toxin  of  the 
disease  for  an  animal  of  such  weight).  Inoculate  this  and  an  unpro- 
tected guinea-pig  each  with  o.i  cubic  centimeter  of  a  bouillon  culture 
of  Mycobacterium  diphtheria  grown  at  body  temperature  for  twenty-four 
or  forty-eight  hours.  Compare  the  results.  From  the  pig  dead  from  the 
inoculation,  at  autopsy  make  smears  from  the  site  of  inoculation  and 
from  the  interior  organs  and  blood.  Stain  these  and  determine  the 
location  of  the  germs.  Make  sections  of  the  tissues  and  study  the  struc- 
tural alterations  evident  about  the  site  of  the  inoculation  and  in  the 
kidneys. 


INDEX. 


A. 

Acid,  acetic,  202,  210 
alcohol,  200 

carbolic,  as  a  disinfectant,  44 
nitric,  solution  of,  200 
phenol-sulphonic,  244 
production,  234 
pyrogallic,  160 
Acquired  immunity,  286 
Aerobic  bacteria,   156 
Aero-bioscope,  Sedgwick-Tucker,   130 
Agar,  gelatine-,  92 
glycerine-,  92. 
lactose-litmus-,  94 
peptonized,  90 

Agglutination  phenomenon,  238 
Air  apparatus,  Hesse's,   132 

Sedgwick-Tucker,  130 
collection  and  examination  of,  128 
Alcohol,  acid,  200 

Alcoholic  solutions  of  aniline  dyes,  198 
Alexines,  236,  238 

Alimentary  inoculation  of  animals,  284 
Alkaline  products  of  bacteria,  234 
Alkaloidal  products  of  bacteria,  236 
Amebabacter,  266 
Amebobacteriaceae,  264,  266 
Amphitrichous  flagella,  214 
Anaerobic  bacteria,  156 
jars,   160 
tubes,  160 

Analytic  key  of  bacterial  genera,  268 
Aniline-water  solution,  Ehrlich's,  200 
Animal  inoculation,  alimentary,  284 
autopsy,  286 
care  after,  288 
cerebro-spinal,  284 
hypodermic,  278 
inhalation,  284 
intra-ocular,  282 
intraperitoneal,  282 
intravenous,  280 
selection  of  animals  for,  278 
tissues,  selective  isolation  by,  254 
Antiseptics,  40,  44 
Antiseptic  solutions,  44 

values,  determination  of,  48 


Antitoxins,  236,  288 

Apparatus  for  air  examination,  130 

for  soil  examination  (Fraenkel),  144 
for  water  examination,  134 

Arnold's  steam  sterilizer,  28 

Artificial  immunity,  288 

Atmosphere  in  cultures,  156 

Autoclave,  30 

Autopsy  of  inoculated  animals,  286 


B. 

Bacillus,  208,  214,  264,  270 

coli  communis,  78,  82,  ex.  48,  ex.  50, 

ex.  59,  ex.  62,  ex.  63,  ex.  64,  ex. 

65,  ex.  66,  ex.  68,  ex.  70,  ex.  76,  ex. 

77,  256,  276 
prodigiosus,  ex.  56,  ex.  59,  ex.  62,  ex. 

71 
subtilis,  ex.  46,  ex.  60,  ex.  64,  ex.  66, 

ex.  68,  ex.  79 

tetani,  ex.  37,  ex.  54,  ex.  55,  ex.  78 
typhosus,  78,  ex.  38,  ex.  43,  ex.  50, 

218,  251,  ex.  52,  ex.  59,  ex.  62,  ex. 

64,  ex.  66,  ex.  67,  ex.  68,  ex.  72, 

256,  ex.  77 

Bacteria,  aerobic,   156 
anaerobic,  156 
analytic  key  of  genera,  268 
capsule,  212 
chemical  products,  226 
chromogenic,  226 
classification  of,  262 
collection  of,  122 
colonies  of,  172 

gross  features,  172 
microscopic  features,  180,  188 
counting  of,  162 
cultivation  of,  148 
demonstration  of,  190 
denitrifying,  242 
destruction  of,  18 
distribution  in  nature,  16 
family  classification.   12    262 
flagella,  214 
granules,  212 
grouping  of,  210 


291 


292 


INDEX. 


Bacteria,  identification  of,  268 

inoculation  of,  108 

isolation  of,  246 

liquefying,  88,  288 

measurement  of,  216 

mesophilic,  150 

motility,  218 

nitrifying,  144,  242 

pathogenic  activity  of,  276 

physical  and  chemical  features,  186 

pigment  of,  226 

place  in  nature,  12 

products  of,  224 

psychrophilic,  150 

relations  to  higher  vegetables,  14 
lower  animals,  14 

reproduction  of,  220 

shape  of,  208 

size  of,  216 

staining  of,  194 

structure  of,  212 

thermophilic,  150 
Bacteriacese,  208,  262,  264,  270 
Bacteriology,  definition  of,  14 
Bacterioplasmins,  236 
Bacterioproteins,  236 
Bacterium,  214,  264,  270 

anthracis,  ex.  82 

pneumoniae,  ex.  49 
Beggiatoa,  218,  266 
Beggiatoaceae,  218,  264,  266 
Bichloride  of  mercury,  42 
Blades  as  inoculating  apparatus,  118 
Blood-serum,  94 

collection  of,  94 

distribution  of,  98 

inspissator,  100 

preparation  of,  100 
Blue,  Gabbett's,   198 

Loeffler's,  198 

Unna's  polychrome,  200 
Boiling  in  sterilization,  24 
Boracic  acid,  44 
Borax,  44 

Bottle  for  collecting  water,   140 
Bouillon,  80 

glucose,  80 

lactose,  80 

saccharose,  80 
Bromine,  42 
Bulb,  Sternberg's,  116 

C. 

Capillary  tube  method  of  isolation,  248 
Caps,  rubber,  148 
Capsules  of  bacteria,  212 
staining  of,  214 

Carbohydrate  culture  media,  72,  78 
Carbol-fuchsin,  Ziehl's,  198 
Carbolic  acid,  44 


Carbol-thionine,  200 

Carbonic  oxide  atmosphere  in  anaerobic 

cultures,  160 

Chameleon  reaction  of  Ps.  pyocyanea,  226 
Chemical  and   physical  features  of  bac- 
teria, 186 

solutions  in  disinfection,  40 
sterilization,  principles  of,  42 
Chemotaxis,  220,  ex.  52. 
Chlamidobacteriaceae,  264,  266,  272 
Chlorinated  lime,  44 
Chlorine,  42 
Cholera.     See  Microspira  choleras 

red  reaction,  240 
Chromateaceae,  264,  266 
Chromatic  granules,  212 
Chromatium,  266 
Chromogenic  bacteria,  226 
Cladothrix,  266,  272. 
Classification  of  bacteria,  12,  262 
Claustridium,  208 
Cleanliness,  laboratory,  10 
Cleansing  tubes  and  flasks,  etc.,  58,  64 
Coccaceae,  208,  262,  264,  268 
Coccus,  208 

Cohn's  heating  method  of  isolation,  260 
Collection  of  infectious  material,  122 
Colon  bacillus.     See  Bacillus  coli 
Colonies  of  bacteria,  172 

counting,  162 

gross  features  of,  172 

isolation  of,  246 

minute  features  of,  188 
Conditions  of  bacterial  development,  148 
Copperas,  44 
Corrosive  sublimate,  42 
Cotton  plugs,  60 
Covers  and  slides,  188 
Crenothrix,  266,  272 
Cultural  conditions,  148 
Culture  flasks,  64 
media,  70 

agar,  90 

gelatine-,  92 
glycerine-,  92 
lactose-litmus,  94 

blood-serum,  94 

bouillon,  80 

carbohydrate,  72 

Dunham's  peptone  solution,  94 

Eisner's,  78 

gelatine,  86 

potato-,  78 

glycerine-agar,  92 

glycerine-potato,  78 

Holz's,  78 

inoculation  of,  108 

lactose-litmus-agar,  94 

litmus  milk,  106 

milk,  104 

potato,  72 


INDEX. 


293 


Culture  media,  potato-gelatine,  78 

preservation  of,  106 

proteid,  78 

reaction  of,  70 

rosolic  acid-peptone  solution,  94 

selective  isolation  by,  256 

serum,  blood-,  94 

Loeftler's    glucose-bouillon-, 

104 

Cultures,  aerobic,  158 
anaerobic,  158 
atmosphere  of,  156 

selective  isolation  by,  258 
capillary  tube,  163,  248 
deep,  124 
diffusion,  124 
Esmarch's  tube,  128 
flask,  128 

gross  features  of,  172 
hanging-drop,  188 
light  for,  160 
minute  features  of,  188 
mixed,  58 
moisture  of,  148 
nutrients  of,  148 
Petri  dish,  126 
plate,  124 
potato  dish,  74 

tube,  76 
pure,  58 
rolled  tube,  128 
smear,   124 
stab,  124 
stroke,  124 
surface,   1 24 
temperature  of,  150 

selective  isolation  by,  258 
tube,  124 

D. 

Death-point,  thermal,  24,  32 
Denitrifying  bacteria,  242 
Diastasic  fermentation,  230 
Diffusion  cultures,  124 

inoculations,  110,  124 
Dilution  inoculations,  120 

isolation,  252 

Diphtheria.     See      Mycobacterium     diph- 
theria; 

Diplococcus,  212,  220 
Disease  material,  examination  of,  144 

production  by  bacteria,  284 
Dish  cultures,  126 
Dishes,  plate  and  potato,  66 

Petri,  66 
Disinfectant  gases,  54 

solution,  42 

values,  determination  of,  46 
Disinfectants,  chemical,  40 
Disinfecting  jars,  10 
Disinfection,  failures  of,  40,  50 


Disinfection,  surgical,  52 
Distribution  flasks,  64 

of  media,  84 
Dry-air  oven  for  sterilization,  20 

E. 

Ehrlich's  aniline  oil- water  solution,  200 
Eisner's  medium,  78 
Enumeration  of  bacteria,  162 
Enzymes,  228 
Esmarch's  tubes,  128 
Essential  oils  as  antiseptic  agents,  44 
Eubacteriaceae,  262,  264,  268 
Evolutional  forms  of  bacteria,  208 
Examination  of  air,   130 

of  inoculated  animal,    post-mortem, 
284 

of  milk,  142 

of  pathologic  material,  144 

of  soil,  144 

of  water,  134 

F. 

Feces,  disinfection  of,  44 
Fermentation,  diastasic,  230 

invertin,  230 

products,  228 

proteolytic,  228 

rennet,  230 

tubes,  64 

urea,  230 

Ferrous  sulphate,  44 
Film,  impression,  194 

preparations,  192 
Filters,  collecting,  136 

porcelain,  36 

Filtration,  sterilization  by,  36 
Fission  of  bacteria,  220 
Fixation,  192,  194,  206 
Flagella,  214 

staining  of,  216 
Flaming,  sterilization  by,  18 
Flask  cultures,  128 
Flasks,  culture-,  64 

distribution-,  64 
Forceps  in  inoculation,  120 

in  staining,  192 
Formaldehyde  as  a  disinfectant,  44,  54 

generators,  54 
Fractional  inoculation,  120 

isolation,  252 

sterilization,  22 
Fraenkel's  soil  harpoon,  144 

G. 

Gabbett's  blue  solution,  198 
Gas  pressure  regulator,  156 

production  by  bacteria,  232 
Gases,  disinfectant,  54 


294 


INDEX 


Gelatine,  86 
Gelatine-agar,  93 
Genera,  analytic  key  of,  268 
Generators,  formaldehyde,  54 
Generic  names,  synonyms  of,  272 
Germicides,  40 
Germination  of  spores,  222 
Glanders,  254 
Glucose-bouillon,  82 
Glucose-bouillon-serum,   104 
Glycerine  agar,  92 

potato,  78 
Gonidia,  224 

Gonorrhea.      See  Micrococcus  gonorrhoea 
Gram's  differential  solution,  200 

method,  202 
Granules,  metachromatic,  212 

nuclear,  212 

staining  of,  214 
Gross  examination  of  bacterial  cultures, 

172 
Grouping  of  bacteria,  210 

H. 

Hands,  disinfection  of,  ex.  19  ^ 
Hanging-drop  preparation,  188 
Heat,  sterilization  by,   18 
Heating  method  of  isolation,  Cohn's,  260 
Hesse's  air  apparatus,  132 
Holz's  medium,  78 
House  sterilization,  54 
Hydrogen  atmosphere  in  anaerobic  cul- 
tures,  160 

disulphide  in  anaerobic  cultures,  160 
Hypodermic  inoculation,  278 

I. 

Identification  of  bacteria,  262 
Immunity,  acquired  and  natural,  286 

active  and  passive,  286 
Impression  films,  194 
Incubator,  152 

temperature,  150 
Indol  production,  240 
Inhalation  inoculation,  284 
Inoculation,  animal,  278 
alimentary,  284 
cerebrospinal,  284 
hypodermic,  278 
inhalation,  284 
intra-ocular,  282 
intraperitoneal,  282 
intravenous,  280 
care  of  animals  after,  284 
needle,  108 
of  media,  108 

by  blades,  118 
by  forceps,  118 
by  particles,  118 


Inoculation  of  media  by  pipettes,  114 

by  Sternberg  bulb,  116 

by  swab,  112 

by  syringe,  116 

diffusion,  110 

dilution,  120 

fractional,  120 

puncture,  110 

smear,  110 

stab,  110 

stroke,  110 
preventive,  288 
selection  of  animals  for,  278 
Insects,  bacteria  carried  by,  130 
Inspissator,  serum,  100 
Intra-ocular  inoculation,  282 
Intraperitoneal  inoculation,  282 
Intravenous  inoculation,  282 
Invertin  fermentation,  230 
Involution  forms  of  bacteria,  208 
Iodide  of  mercury,  44 
Iodine,  42 
lodoform,  44 
Isolation  of  bacteria,  246 

by  capillary  tubes,  248 

by  needle,  248 

Cohn's  heating  method,  260 

dilution  methods,  252 

plating  methods,  246 

selection  by  animal  tissues,  254 
by   atmosphere   of   culture, 

258 

by  medium  of  culture,  256 
by  temperature  of  culture, 
258 


Jars,  anaerobic,  160 
disinfecting,  1 0 
preserving,  1 06 

K. 

Key  of  bacterial  genera,  analytic,  268 
Kitasato's  filter,  36 

Klebs'  fractional  isolation  method,  252 
Koch's  postulates,  146,  246,  278,  286 
steam  sterilizer,  28 

L. 

Labelling  cultures,  12,  58 
Laboratory  cleanliness,  10 

provisions,  10 
Lactose  bouillon,  82 
Lactose-litmus-agar,  94 
Lamprocystaceae,  264,  266 
Lamprocystis,  266 
Leptothrix,  266,  272 
Lethal  exposure,  thermic,  24,  32 


INDEX 


295 


Light,  influence  on  bacteria,  162 

reaction  of  bacteria,  226 
Litmus  milk,  106 

Loeffler's  glucose-bouillon  serum,  104 
Lm     methylene-blue  solution,   198 

staining  with,  202 
Loop,  inoculating,  108 
Lophotrichous  flagella,  214 

M. 

Matlock's  filtration  buckets,  92 
Measurement  of  bacteria,  216 
Mechanical  methods  of  isolation  of  bac- 
teria, 216 
Media,  culture,  70 
agar,  90 
blood-serum,  94 
bouillon,  80 
carbohydrate,   72,   78 
distribution  of,  84 
Eisner's,  78 

for  nitrifying  bacteria,  144 
for  phosphorescent  bacteria,  228 
gelatine,  86 
gelatine-agar,  92 
glycerine  potato,  78 
Holz's,  78 
inoculation  of,  108 
lactose-litmus  agar,  94 
litmus  milk,  106 
milk,  104 

peptone  solution,  94 
potatoes,  72 
preservation  of,  106 
proteid,  78 
reaction  of,  70 

rosolic  acid-peptone  solution,  94 
Mercuric   albuminates   found  in  disinfec- 
tion, 40,  ex.  18 
chloride,  42 
iodide,  44 
nitrate,  44 

Mesophilic  bacteria,   150 
Metachromatic  granules,  212 
Micrococcus,  212,  264,  268 
gonorrhoea?,  ex.  42 
pyogenes,  ex.  40,  ex.  41,  ex.  51,  ex. 
52,  ex.  59,  ex.  62,  ex.  64,  ex.  66,  ex. 
68,  ex.  71,  ex.  81 
tetragenus,  ex.  47,  ex.  49 
Microscope,  186 
Microscopic  examination  of  colonies,  188 

of  individual  bacteria,  190 
Microspira,  214,  264,  270 

cholerae.      See  M.  comma 

comma,  ex.  44,  ex.  50,  ex.  59,  ex.  61, 

ex.  62,  ex.  64,  ex.  66,  ex.  69,  276 
Milk  as  a  culture  medium,  104 
examination  of,  142 
litmus-,  106 


Milk   of  lime,  44 

Pasteurization    and    sterilization    of, 

34,  ex.  12 
purity  of,   142 
Moisture  of  cultures,  148 
Monotrichous  flagellation,  214 
Mordants  in  staining,  196 
Motility  of  bacteria,  218 

determination  of,  220 
Multiplication  of  bacteria,  220 
Murrell  gas  pressure  regulator,  156 
Mustard  oil,  44 

Mycobacteriaceae,  14,  208,  264,  266,  270 
Mycobacterium,  208,  266,  270 

diphtherias,  104,  120,  ex.  36,  ex.  51, 
ex.  59,  ex.  62,  ex.  64,  ex.  66,  ex.  68, 
252,  ex.  83 
tuberculosis,  204,  ex.  47,  ex.  51,  ex.  75 


N. 

Names,  synonymous  generic,  272 

Needle,  inoculating,  108 

Nitric  acid  solution,  200 

Nitrifying  bacteria,  144,  242 

Nitrogen  atmosphere  in  anaerobic  cultures, 

158 

Nitroso-indol  reaction,  240 
Non-pathogenic  bacteria,  14 
Nuclear  granules,  212 
Nutrients,  cultural,  148 


O. 

Obligate  aerobic  bacteria,  156 

anaerobic  bacteria,  156 

parasitic  bacteria,  148 
Oese,   108 
Oils,  essential,  44 
Oligomorphism,  208 
Oven,  dry-air  sterilizing,  20 
Oxygen,  influence  on  bacteria,  156 

P. 

Paraform,  54 

Parasitic  bacteria,  14,  148,  276 

Particles  in  inoculation,   118 

Passive  immunity,  288 

Pasteurization,  34 

Pathogenic  activity  of  bacteria,  276,  284 

bacteria,   14,  276 

culture  of,   142,   144 

Pathologic  material,  examination  of,   144 
Peptone  solution,  Dunham's,  94 
Peptonized  agar-agar,  90 

gelatine,  86 

Peritrichous  flagella,  214 
Permanganate  of  potassium,  44 
Petri  dish  cultures,  126 

dishes,  66 


296 


INDEX 


Phenol,  240 

Phenol-sulphonic  acid,  244 

Photogenic  activity,  226 

Phragmidiothrix,  244,  266 

Physical  and  chemical  characteristics  of 

bacteria,   186 

Physiologic  methods  of  isolation,  254 
Pigment  production,  226 
Pipettes,  capillary,  114 

for  inoculation,  114 
Plan  of  work,  9 
Planococcus,  214,  264,  270 

agilis,  ex.  50 

Planosarcina,  214,  264,  270 
Plate  and  potato  dishes,  66 

cultures,  124 
Plates,  glass,  68 
Plating  a  tube,  126 

methods,  246 
Platinum  needle,  108 
Pleomorphism,  208 
Plugs,  cotton,  60 
Point,  thermal  death-,  32 
Polychromic  blue  solution,  Unna's,  200 
Porcelain  niters,  36 
Post-mortem   examination  of  inoculated 

animal,  286 
Potato  as  a  culture  medium,  72 

glycerine-,  78 

paste,  76 

tubes,  60 

Potato-gelatine,  78 
Preservation  of  media,  106 
Pressure  regulator,  gas,  156 
Preventive  inoculation,  288 
Production  of  disease  by  bacteria,  284 
Products  of  bacteria,  226 
Proteid  culture  media,  78 
Proteolytic  ferment,  228 
Provisions,  laboratory,  10 

personal,  12 
Pseudomonas,  214,  264,  270 

pyocyanea,  ex.  39,  ex.  50,  ex.  57,  ex. 

72 

chameleon,  reaction  of,  226 
Psychrophilic  bacteria,  150 
Ptomaines,  236 
Puncture  inoculations,  110 
Pure  cultures,  isolation  of,  246 
Pyrogallic  acid,  160 

R. 

Reaction,  chameleon,  226 

cholera  red,  240 

of  culture  media,  70 
Reagents,  staining,  194 

sterilizing,  42,  54 
Regulator,  gas  pressure,  156 

temperature,    154 
Rennet  ferment,  230 


Reproduction  of  bacteria,  220 
Rhabdochromatium,  268 
Rhodobacteriaceae,  226,  264,  266 
Rolled  tubes,  Esmarcl     ,128 
Rooms,  disinfection  of,  54 
Rosolic  acid-peptone  solution,  94 
Rotting,  16 

Rubber  caps  for  tubes,  106 
stoppers  for  tubes,  106 
Rules  of  Koch,  146,  246,  278,  286 


S. 

Saccharose-bouillon,  82 

Salomonsen's  capillary  tubes,  248 

Saprophytes,  14 

Sarcina,  210,  212,  220,  264,  270 

Schizomycetes,  264 

Sealing  test-tubes,  106 

Sections  of  tissues,  staining,  206 

Sedgwick-Tucker  aero-bioscope,  130 

Selective  isolation  of  bacteria  by  animal 

tissues,  254 
by  Cohn's  heating  methods, 

260 

by  culture  atmosphere,  258 
medium,  256 
temperature,  258 

Serum,  agglutination  phenomenon  of,  238 
blood-,  94 
collection  of,  94 
distribution  of,  98 
glucose-bouillon,    104 
inspissator,  100 
Shape  of  bacteria,  208 
Size  of  bacteria,  216 
Slides  and  covers,  188 
Smear  inoculations,  110 
Soil,  bacteria  in,  144 

examination  of,  144 
Fraenkel's  harpoon  for  collecting,  144 
Solution,  antiseptic,  44 
disinfectant,  42 
Gram's,  200 
of  acetic  acid,  202 
of  aniline  dyes,  198 

oil,  200 

of  carbolic  acid,  44 
of  carbol-fuchsin,  200 
of  carbol-thionine,  200 
of  chlorinated  lime,  44 
of  copperas,  44 
of  formaldehyde,  44 
of  lime,  44 

of  mercuric  chloride,  42 
iodide,  42 
nitrate,  42 
of  methylene-blue,  Gabbett's,   198 

Loeffler's,  198 
of  nitric  acid,  200 
of  peptone,  Dunham's,  94 


INDEX. 


297 


Solution  of  polychromic  blue,  Unna's,  200 

of  rosolic  acid-peptone,  94 

staining,   198 
Spirillaceae,  208/  J64,  270 
Spirillum,  208,  214,  264,  270 
Spirochseta,  212,  264,  270 
Spirosoma,  214,  264,  270 
Spores,  determination  of,  32,  224 

germination  of,  222 

staining  of,  222 
Sporulation,  222 

Sputum,  staining  of,  tuberculous,  204 
Stab  inoculations,  110 
Staining,  Gram's  method,  202 

Loeffler's  method,  202 

methods,  196,  202 

mycobacterium  of  tuberculosis,  204 

nuclear  granules,  214 

of  capsules,  214 

of  flagella,  216 

sections,  206 

spores,  222 

sputum  of  tuberculous  patients,  204 

to  show  grouping,  of  bacteria,  210 
Staphylococcus,  212 
Steam  sterilization,  26 

sterilizers,  26,  28,  30 
Sterilization  and  Pasteurization,  34 

by  autoclave,  30 

by  boiling,  24 

by  chemical  gases,  54 
solutions,  40 

by  dry-air  oven,  20 

by  nitration,  36 

by  naming,  18 

by  heat,   18 

by  steam,  24,  26,  28 

by  Tyndall's  method,  22 

definition  of,  18 

fractional,  22 

of  clothes,  24,  30,  54 

of  dishes,  66 

of  feces,  44 

of  flasks,  64 

of  plates,  68 

of  rubber  goods,  42 

of  tubes,  62 

Sterilizers,  steam,  Arnold's,  28 
autoclave,  30 
Koch's,  26 
Sternberg's  bulbs,  116 

method   of   determining   disinfectant 

values,  46 

Stewart's  forceps,  192 
Stools,  disinfection  of,  44 
Stoppers,  cotton,  36,  60 

rubber,  148 
Streptobacilli,  212 
Streptococcus,  212,  220,  264,  268 
Streptothrix,  266,  272 
Stroke  cultures,  110 
20 


Structure  of  bacteria,  212 

Sulphanilic  acid,  242 

Sulphur  dioxide,  54 

Sunlight,  162,  170 

sSynonyms  of  generic  names,  272 

Syringes,  116 

T. 

Temperature,  cultural,  150 

incubator,  150 

maximum,  150 

minimum,  150 

optimum,  150 

regulator,  152 
Test-tubes,  58 

anaerobic,   160 

cleansing,   58 

marking,  12,  58 

plugging,  60 

sterilizing,  62 

Tetanus.       See  Bacillus  tetani 
Tetrads,  212,  220 
Thermal  death-point,  32 
Thermophilic  bacteria,  150 
Thermostat,   152 
Thiobacteriaceae,  226,  262,  266 
Thiocapsa,  266 
Thiocapsaceae,  264,  766 
Thiocystis,  266 
Thiodictyon,  266 

Thionine,  carbolized  solution  of,  200 
Thiopedaceae,  266 
Thiopedia,  266 
Thiopolycoccus,  266 
Thiosarcina,  266 
Thiospirillum,  268 
Thiothece,  266 
Thiothrix,  266 
Toxalbumins,  236 
Toxins,  236 
Tube  cultures,  124 

Esmarch's  rolled,  128 
Salomonsen's  capillary,  248 
Tubes,  capillary,  248 

caps  of,  148 

cleansing  of,  58 

fermentation,  64 

plugging,  60 

potato,  60 

sterilizing,  62 

of  rubber,  42 

Tyndall's  fractional  method  of  steriliza- 
tion, 22 
Typhoid  fever.       See  Bacillus  typhosus 


U. 

Unna's  polychrome  blue,  200 
Urea  fermentation,  230 


298 


INDEX. 


Vaccination,  288 


V. 


w. 


Water,  bacteria  of,  134 

collection  and  examination  of,  134 

bottle,   140 

filter,  136 

pathogenic  bacteria  in,  142 
purity  of,   134 


Watery  solutions  of  aniline  dyes,  194 
Widal's    agglutination    test    in    typhoid 

fever,  238 
Wire  basket,  60 
platinum,  108 

Z. 

Ziehl's  carbol-fuchsin  solution,  198 
Zooglea,  210,  220 
Zymose,  234 


ERRATA. 

The  legend  for  Fig.  18  should  read  "Culture  Dish." 

In  legend  of  Fig.  40,  and  third  line  below,  read  for  "  Sedgwick-Turner,"  "  Sedgwick-Tucker." 


For   Fig.    66,    substitute 


FIG.  66. — POLAR,  BIPOLAR, 
AND  EQUATORIAL  GERMI- 
NATION OF  SPORES. 


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Catalogue  No.  9. — Books  on  Nervous  and  Mental  Diseases. 

Catalogue  No.  10. — Books  on  Diseases  of  the  Eye,  Refraction,  Spectacles,  etc. 

A  Genera.1  Catalogue. — Containing  2000  titles  of  Standard  Books  on  Medicine  and  Sur- 
gery. American  and  English. 

Special  Circulars. — Morris'  Anatomy;  Gould  and  Pyle's  Cyclopedia;  Deaver's  Surgical 
Anatomy;  Tyson's  Practice;  Gould's  Medical  Dictionaries;  Books  on 
the  Eye ;  Books  on  Diseases  of  the  Nervous  System  ;  The  ?  Quiz- 
Compend?  Series,  Visiting  Lists,  etc.  We  can  also  furnish  sample 
pages  of  most  of  our  publications. 


P.  Blakiston's  Son  &  Co.'s  publications  may  be  had  through  the  booksellers  in  all 
the  principal  cities  of  the  United  States  and  Canada,  or  any  book  will  be  sent,  postpaid,  upon  receipt 
of  the  price.  Special  terms  of  payment  will  be  allowed  to  those  of  approved  credit.  No  discount 
can  be  allowed  retail  purchasers  under  any  circumstances.  Money  should  be  remitted  by  express 
or  post-office  money  order,  registered  letter,  or  bank  draft. 


THE  PRICES  OF  ALL  BOOKS  ARE  NET. 


CLASSIFIED  LIST,  WITH  PRICES, 

OF  ALL  BOOKS  PUBLISHED  BY 
P.  BLAKISTON'S  SON  &  CO.,  PHILADELPHIA. 

When  the  price  is  not  given  below,  the  book  is  out  of  print  or  about  to  be  published. 
Cloth  binding,  unless  otherwise  specified.     For  full  descriptions  see  following  Catalogue. 


ANATOMY. 

Ballou.     Veterinary  Anat.     $0.80 
Broomell.       Anatomy     and 

Histol.  of  Mouth  and  Teeth.  4.50 
Campbell.       Dissection    Out- 
lines.         .50 

Deaver.     Surgical  Anatomy.  21.00 
Gordinier.  Anatomy  of  Nerv- 
ous System.     Illustrated.        6.00 
Heath.    Practical.     7th  Ed.      4.25 
Holden.  Dissector.    2  Vols.     3  oo 

Osteology.     8th  Ed.      5.25 

Landmarks.     4th  Ed.      .75 

Hughes.     Dissector.    Part  I.  3.00 

Part  II.  -        -        3.00 

Part  III.         -        -        3.00 

Macalister's  Text-Book.     -    5.00 

McMurrich.     Embryology.    

Minot.     Embryology.        -        

Marshall's   Phys.  &  Anatom- 
ical Diagrams.     $40.00  and  60.00 
Morris.  Text-Book  Anat.  New 

Ed.  846  111.    Clo.,$6.oo;  Sh.,  7.00 
"Potter.      Compend  of.       6th 

Ed.     133  Illustrations.  -  .80 

Wilson's  Anatomy,  nth  Ed.  5.00 

ANESTHETICS. 
Buxton.    Anesthetics.    -         1.50 
Turnbull.    4th  Ed.          -         2.50 

BACTERIOLOGY. 
Conn.     Agricultural    Bacteri- 
ology.   Illustrated.      -      -     2.50 

Bact.  in  Milk  Products. 

Emery.     Bacteriolog.  Diag.     1.50 
Hewlett.    Manual  of.    lllus.     4.00 
Williams.     Student's  Manual 

of.     2d  Edition,     oo  lllus.       1.50 

BRAIN  AND  INSANITY. 
Blackburn.     Autopsies.    -      1.25 
Chase.     General  Paresis.          1.75 
Horsley.  Brain  and  Sp.  Cord.    2.50 
Ireland.     Mental    Affections 

of  Children.  ...  4.00 
Lewis.  Mental  Diseases.  7.00 
Mann's  Psychological  Med.  3.00 
Regis.  Mental  Medicine.  -  2.00 
Stearns.  Mental  Dis.  lllus.  2.75 
Tuke.  Dictionary  of  Psycho- 
logical Medicine.  2  Vols.  10.00 
Wood.  Brain  and  Overwork.  .40 

CHEMISTRY. 

Technol'g'l  Books,  Water, Milk, etc, 
Allen.     Commercial    Organic 
Analysis.     Vol.  I.       -        -    4.50 

Vol.  II.     Part  I.       -    3.50 

Vol.11.     Part  II.      -    3.50 

Vol.11.     Part  III.    -    

Vol.  III.     Part  I.     -     4.50 

Vol.  III.     Part  II.    -    4.50 

Vol.  III.    Part  III.  -    4-50 

Vol.  IV.      -        -      -    4.50 

Bailey    and    Cady.      Chein. 

Analysis.-        -        -        -         1.25 
Bartley.    Medical  Chemistry.  3.00 

Clinical  Chemistry.        i.oo 

Bloxam's  Text-Book,  gth  Ed. 

Bunge.  Physiologic  and  Path- 
ologic Chemistry.     -        -        3.00 

Caldwell.      Qualitative    and 

Quantitative  Analysis.        -     i.co 
Cameron.    Oils  &  Varnishes.  2.25 

Soap  and  Candles.     -    2.00 

Clowes  and  Coleman.  Quan- 
titative Analysis,    sth  Ed.  -  3.50 

Coblentz.  Volumetric  Anal.  1.25 
Congdon.  Laboratory.  -  i.oo 
Gardner.  Brewing,  etc.  -  1.50 
Gray.  Physics.  Vol.  I.  -  4.50 
Groves  and  Thorp.  Chemi- 
cal Technology,  vol.  I.  Fuels  5.00 

. Vol.  II.     Lighting.      -     4.00 

. Vol.  III.  Gas  Lighting.  3  50 

Vol.  IV.  Elec.  Lighting. 

Heusler.     The  Terpenes.         4.00 


Holland.  Urine,  Gastric  Con- 
tents, Poisons  and  Milk  Anal- 
ysis. 6th  Ed.  -  -  -  $1.00 

Leffmann's  Medical  Chem.        .80 

Food  Analysis.      -      -     2.50 

Milk  Analysis.     -      -      1.25 

Water  Analysis.       -         1.25 

Structural  Formulae.        i.oo 

Muter.     Pract.  and  Anal.         1.25 
Oettel.     Electro-Chem.          -     .75 
— — —  Electro-Chem.  Exper.-      .75 
Richter's  Inorganic,   sth  Ed.   1.75 
Organic.  3d  Ed.  2  Vols. 

Vol.  I.  Aliphatic  Series.    3.00 
Vol.11.  Carbocyclic  "        3.00 
Rockwood.     Chemical  Anal.  1.50 
Smith. Electro-Chem.  Anal.     1.25 
Smith  and  Keller.     Experi- 
ments.    4th  Ed.     lllus.  .60 
Sutton.     Volumetric  Anal.        5.00 
Symonds.     Manual  of.             2.00 
Traube.  Physico-Chem.Meth.  1.50 
Thresh.     Water  Supplies.   -    2.00 
Ulzer  and  Fraenkel.    Tech- 
nical Chemical  Analysis.          1.25 
Woody.  Essentials  of.  4th  Ed.  1.50 

CHILDREN. 

Hatfield.     Compend  of.  .80 

Power.  Surgical  Diseases  of.  2.50 
Smith.  Wasting  Diseases  of.  2.00 
Starr.  Digestive  Organs  of.  3.00 

Hygiene  of  the  Nursery.i.oo 

Taylor  and  Wells.  Manual.  4.50 

CLINICAL  CHARTS,  ETC. 
Griffith's     Temp't're  Charts. 

Pads  of  50  -        .          -  .50 

Keen.     Outline    Drawings    of 

Human  Body.     Pads.    -  .25 

Schreiner.   Diet  Lists.   Pads,   .75 

COMPENDS. 

Ballou.     Veterinary  Anat.  .80 

Brubaker's  Physiol.  zoth  Ed.  .80 

Gushing.     Histology.     -    -  .80 

Gould  and  Pyle.    The  Eye.  .80 

Hatfield.     Children.          -  .80 

Horwitz.    Surgery,     sth  Ed.  .80 

Hughes.  Practice.  2  Pts.  Ea.  .80 

Landis.    Obstetrics.    7th  Ed.  .80 

Leffmann's  Chemistry.  4th  Ed.  .80 

Potter's    Anatomy,   6th  Ed.  .80 

Materia  Medica.  6th  Ed.  .80 

Schamberg.     Skin  Diseases.  .80 

Stewart.  Pharmacy,  sth  Ed.  .80 

Thayer.     General  Pathology.  .80 

Special  Pathology.  .80 

Warren.     Dentistry,    sd  Ed  .80 

'Wells.     Gynecology.    2d  Ed.  .80 

Any  of  above,  Interleaved,  $1.00. 
Self-  Examination.         3500 
Questions   on   Medical    Sub- 
jects.         -          -          Paper,   .10 

CONSUMPTION. 
Knopf.  Pulmon. Tuberculosis.  3.00 
Steell.    Physical  Signs  of  Pul- 
monary Disease.      -      -      -     1.25 

DENTISTRY. 

Barrett.  Dental  Surg.  -  i.oo 
Broomell.  Anat.  and  Hist,  of 

Mouth  and  Teeth.  -  -  4.50 
Fillebrown.  Op.  Dent.  lllus.  2.25 
Gorgas.  Dental  Medicine.  4.00 

Questions  and  Answers.  6.00 

Harris.  Principles  and  Prac.  6.00 
— — —  Dictionary  of.  6th  Ed.  5.00 
Richardson.  Mech.  Dent.  5.00 

Smith.     Dental  Metallurgy.    

Taft.  Index  of  Dental  Lit.  2.00 
Tomes.  Dental  Surgery.  4.00 

Dental  Anatomy.  4.00 

Warren's  Compend  of.    -          .80 


Dental    Prosthesis  and 
Metallurgy.     Ilh 


Metallurgy,     lllus. 
White.    Mouth  and  Teeth. 


DIAGNOSIS. 

Brown.     Medical.    4th  Ed.  $2.25 

Tyson's  Manual.  4th  Ed.  lllus.  1.50 
DICTIONARIES,  ETC. 

Gould's  Illustrated  Dictionary 
of  Medicine,  Biology,  and  Al- 
lied Sciences,  etc.  sth  Edi- 
tion. Leather,  $10.00;  Half 
Russia,  Thumb  Index,  -  12  oo 

Gould'sStudent's  Medical  Dic- 
tionary, nth  Ed.,  lllus.  ,*4 
Mor.,  $2.50;  Thumb  Ind.,  3.00 

Gould's  Pocket  Dictionary — 
30,000  medical  words.  4th 
Edition.  Enlarged.  Leather,  i.oo 

Gould  and  Pyle.  -Cyclopedia 
of  Med.  and  Surg.  One  Vol. 
lllus.  Leather,  10.00 

Gould  and  Pyle's  Pocket 
Cyclopedia  of  Medicine.  i.oo 

Harris'  Dental.  Clo.  5.00;  Shp.  6.co 

Longley's  Pronouncing.  .75 

Maxwell.  Terminologia Med- 
ica Polyglotta.  -  -  3.00 

Treves.     German-English.        3.25 

EAR. 

Burnett.     Hearing,  etc.  .40 

Hovell.     Treatise  on.  -     5.50 

Pritchard.  Diseasesof.  3d  Ed.  1.50 

ELECTRICITY. 

Bigelow.  Plain  Talks  on.  i.oo 
Hedley.  Therapeutic  Elec.  2.50 
Jacobi.  Electrotherapy.  2Vols.  5.00 
Jones.  Medical  Electricity.  3.00 

EYE. 

Donders.  Refraction.  -  1.25 
Fick.  Diseases  of  the  Eye.  4.50 
Gould  and  Pyle.  Compend.  .80 
Greeff.  Microscopic  Examin- 
ation of.  -  -  -  -  1.25 
Harlan.  Eyesight.  -  .40 

Hartridge. Refraction. nth  Ed. i. 50 

Ophthalmoscope.4th  Ed.  1.50 

Hansell   and    Reber.     Mus- 
cular Anomalies  of  the  Eye.  1.50 
Hansell  and   Bell.    Clinical 

Ophthalmology.  120  lllus.  1.50 
Jennings.  Ophthalmoscopy.  1.50 
Morton.  Refraction.  6th  Ed.  i.oo 
Ohlemann.  Ocular  Therap.  1.75 
Parsons.  Optics.  -  2.00 

Phillips.   Spectacles  and  Eye- 
glasses.    49  lllus.     2d  Ed.      i.oo 
Swanzy's  Handbook.  7th  Ed.  2.50 
Thorington.     Retinoscopy.      i.oo 

Refraction.     200  lllus.  1.50 

Walker.  Student's  Aid.  1.50 
Wright.  Ophthalmology.  3.00 

GYNECOLOGY. 

Bishop.  Uterine  Fibromyo- 
mata.  Illustrated.  -  -  3.50 

Byford  (H.  T.).  Manual.  3d 
Edition.  363  Illustrations.  3.00 

Duhrssen.     Gynecological 

Practice.     105  Illustrations.    1.50 

Lowers.     Dis.  of  Women.        2.50 

Montgomery.  Text -book 
of.  527  lllus.  ...  5.00 

Roberts.  Gynecological  Path- 
ology. Illustrated.  •  6.00 

Wells.    Compend.     lllus. 


.80 


HEALTH  AND  DOMESTIC 

MEDICINE. 

Bulkley.    The  Skin.        -  .40 

Burnett.     Hearing.          -  .40 

Cohen.     Throat  and  Voice.  .40 

Dulles.  Emergencies,  sth  Ed.  i.oo 

Harlan.     Eyesight.          -  .40 

Hartshorne.    Our  Homes.  .40 

Osgood.    Dangers  of  Winter.  .40 

Packard.    Sea  Air,  etc.  .40 

Richardson's  Long  Life.  .40 

White.     Mouth  and  Teeth.  .40 
^Vilson.     Summer  and  its  Dis.  .40 


CLASSIFIED  LIST  OF  P.  BLAKISTON'S  SON  6*  CO.'S  PUBLICATIONS. 


HISTOLOGY. 

Gushing.  Compend.  -  -  $0.80 
Stirling.  Histology,  ad  Ed.  2.00 
Stohr's  Histology.  Illus.  -  3.00 

HYGIENE. 

Canfield.  Hygiene  of  the  Sick - 
Room.      ....        1.25 

Coplin.     Practical  Hygiene.    

Kenwood.      Public     Health 

Laboratory  Guide.  -  2.00 
Lincoln.  School  Hygiene.  .40 
McFarland.  Prophylaxis.  2.50 
Notter.  Practical  Hygiene.  7.00 
Parkes'  (L.  C.),  Manual.  3  oo 

Elements  of  Health.        1.25 

Rosenau.     Disinfection  and 

Disinfectants.      Illus.         -      2.00 
Starr.  Hygiene  of  the  Nursery,  i.oo 
Stevenson  and  Murphy.  A 
Treatise  on  Hygiene.     In  3 
Vols.     Circular          Vol.  I,   6.00 
upon  application.      Vol.  II,  6.00 
Vol.  Ill,   5.00 

Thresh.  Water  Supplies.  a.oo 
Wilson's  Handbook.  8th  Ed.  3.00 
Weyl.  Coal-Tar  Colors,  1.25 

MASSAGE,  ETC. 

Mitchell  and  Gulick.     Me- 

chanotherapy.  Illustrated.  2.50 
Ostrorn.  Massage.  115  Illus.  i.oo 

MATERIA  MEDICA. 

Biddle.   isth  Ed.    Cloth,         

Bracken.  Materia  Med.  2.75 
Coblentz.  Newer  Remedies,  i.oo 
Davis.  Essentials  of.  -  1.50 
Gorgas.  Dental,  sth  Ed.  4.00 
Grorf.  Mat.  Med.  for  Nurses.  1.15 
Heller.  Essentials  of.  -  1.50 
Potter's  Compend  of.  6th  Ed.  .80 
Potter's  Handbook  of.  gth 

Ed.  Cloth,  $5. oo ;  Sheep,  6.00 
Sayre.  Organic  Materia  Med. 

and  Pharmacognosy.  -  4.50 
Tavera.  Medicinal  Plants  of 

the  Philippines.        -        -        2.00 
White  and  Wilcox.      Mat. 
Med.,  Pharmacy,  Pharmacol- 
ogy, and  Therapeutics,    sth 
Ed.  Enlarged.  Cl., $3.00;  Sh.  3.50 

MEDICAL  JURISPRUDENCE. 

Mann.     Forensic  Med.        -     6.50 
Reese.  Med.  Jurisprudence  and 
Toxicology. sth  Ed.  $3.00;  Sh.  3.50 

MICROSCOPE. 
Carpenter.    The  Microscope. 

Sth  Ed.     850  Illus.  -         8.00 

Lee.  Vade  Mecum  of.  sth  Ed.  4.00 

Oertel.     Med.  Microscopy.     

Reeves.  Med.  Microscopy.      2.50 
Wethered.     Medical  Micros- 
copy.    Illus.       ...     2.00 

MISCELLANEOUS. 
Black.     Micro-organisms.  .75 

Burnet.  Food  and  Dietaries.  1.50 
Cohen.  Organotherapy.  2.50 

Da  Costa.  Hematology.  -  5.00 
Davis.  Alimentotherapy.  2.50 
Goodall  and  Washbourn. 

Infectious  Diseases.  Illus.  3.00 
Gould.  Borderland  Studies.  2.00 
Greene.  Medical  Examination 

in  Life  Insurance.  Illus.  •  4.00 
Haig.  Uric  Acid,  sth  Ed.  3.0* 

Diet  and  Food.  3d.  Ed.  i.oo 

Hare.     Mediastinal  Disease.    2.00 
Hemmeter.  Diseases  of  Stom- 
ach.    2d  Edition.    Illus.    -    6.00 

Diseases  of  Intestines. 

Illustrated.     2  Vols.       -        10.00 

Henry.  Anaemia.  -  -  .50 
McCook.  Amer.  Spiders.  40.00 
New  Sydenham  Society's 

Publications,  each  year.  -  8.00 
Schofield.  The  Force  of  Mind.  2.00 
Thome.  Schott  Methods  in 

Heart  Disease.  -  -  2.00 
Tiasier.  Pneumatotherapy.  2.50 
Treves.  Physical  Education.  .75 
Weber  and  Hinsdale.  Cli- 
mate. 2  Vols.  Illustrated.  5.00 
Winternitz.  Hydrotherapy.  2.50 


NERVOUS  DISEASES,  ETC. 

Dercum.  Rest,  Mental  Thera- 
peutics, Suggestion.  -  $2.50 

Gordinier.  Anatomy  of  Cen- 
tral Nervous  System.  -  6.00 

Gowers.  Manual  of.  530  Illus. 
Vol.  I, $4.00;  Vol.11,  -  4.00 

Syphilis  and  the  Ner- 
vous System.       ...      i.oo 

Epilepsy.    New  Ed.       3.00 

Ormerod.     Manual  of.      -        i.oo 
Pershing.    Diagnosis  of  Nerv. 

and  Mental  Diseases.  -  1.25 
Preston.  Hysteria.  Illus.  2.00 

NURSING. 

Canfield.  Hygiene  of  the  Sick- 

Room.  ....  1.25 
Cuff.  Lectures  on.  3d  Ed.  1.25 
Davis.  Bandaging.  Illus.  1.50 
Domville's  Manual.  Sth  Ed.  .75 
Fullerton.  Obst.  Nursing,  i.oo 

Surgical  Nursing.  i.oo 

Gould.     Pocket  Medical  Dic- 
tionary.    Limp  Morocco.       i.oo 

Groff.  Mat.  Med.  for  Nurses.  1.25 
Hadley.  Manual  of.  -  1.25 
Humphrey.  Manual.  23d  Ed.  i.oo 
Starr.  Hygiene  of  the  Nursery,  i.oo 
Temperature  Charts.  Pads.  .50 
Voswinkel.  Surg.  Nursing,  i.oo 

OBSTETRICS. 
Cazeauxand  Tarnier.  Text- 
Book  of.     Colored  Plates.       4.50 

Edgar.    Text-book  of.         -    

Landis.  Compend.  6th  Ed.  .80 
Winckel's  Text-book.  5.00 

PATHOLOGY. 

Barlow.  General  Pathology.  5.00 
Blackburn.  Autopsies.  1.25 

Coplin.   Manual  of.    3d  Ed.       3.50 
Da  Costa.     The  Blood.       -     5.00 
Roberts.   Gynecological  Path- 
ology.    Illustrated.         -          6.00 
Thayer.     General  Pathology      .80 

Special  Pathology.  .80 

Virchow.     Post-mortems.  .75 

Whitacre.   Lab.  Text-book.    1.50 

PHARMACY. 
Beasley's  Receipt-Book.      -    2.00 

Formulary.      -        -          2.00 

Coblentz.  Manual  of  Pharm.  3.50 
Proctor.  Practical  Pharm.  3.00 
Robinson.  Latin  Grammar  of.  1.75 
Sayre.  Organic  Materia  Med. 

and  Pharmacognosy.  2d  Ed.  4.50 
Scoville.  Compounding.  2.50 
Stewart's  Compend.  $th  Ed.  .80 
U.  S.  Pharmacopoeia.  7th 

Revision.  1890  Cl. $2. 50;  Sh.,  3.00 

Postage  extra,   .27 

Select  Tables  from  U.  S.  P.       .25 

PHYSIOLOGY. 

Birch.  Practical  Physiology.  .1.75 
Brubaker's  Compend.  loth  Ed.  .80 
Jones.     Outlines  of.      -        -     1.50 
Kirkes'  New  i7th  Ed.    (Halli- 
burton.)   Cloth,  $3.00;   Sh.,  3.75 

Landois'  Text-book.  845  Illus. 

Starling.  Elements  of.  -  i.oo 
Stirling.  Practical  Phys.  2.00 
Tyson's  Cell  Doctrine.  -  1.50 

POISONS. 

Reese.  Toxicology.  4th  Ed.  3.00 
Tanner.  Memoranda  of.  .75 

PRACTICE. 

Beale.    Slight  Ailments.  1.25 

Fagge.     Practice.     Vol.  I,       6.00 

Vol.11,    

Fowler's  Dictionary  of.  -  3.00 
Gould  and  Pyle.  Cyclopedia 

of  Medicine.  Illustrated.  10.00 
Hughes.  Compend.  2  Pts.  ea.  .80 
^— —  Physician's  Edition. 

i  Vol.  Morocco,  Gilt  edge.     2.25 
Taylor's  Manual  of.  6th  Ed.    4.00 
Tyson.    The  Practice  of  Medi- 
cine. Illus.  Cl.  $5.50;  Sheep     6.50 


SKIN. 

Bulkley.  The  Skin.  -  $0.40 
Crocker.  Dis.  of  Skin.  Illus.  5.00 
Schamberg.  Compend.  .80 

Van  Harlingen.  Diagnosis 

and  Treatment  of  Skin  Dis. 

3d  Ed.     60  Illus.      -        -        2.75 

SURGERY  AND  SURGICAL 

DISEASES. 

Berry.  Thyroid  Gland.  -  4.00 
Butlin.  Surgery  of  Malignant 

Disease.          -  -  4.50 

Davis.  Bandaging.  Illus.  1.50 
Deaver.  Appendicitis.  -  5.30 

Surgical  Anatomy.     -  21.00 

Dulles.  Emergencies.  -  i.oo 
Hamilton.  Tumors.  3d  Ed.  1.25 
Heath's  Minor.  i2thEd.  1.50 

Clinical  Lectures.        -     200 

Horwitz.  Compend.  sth  Ed.     .80 
Jacobson.     Operations  of.  -   10.00 
Keay.     Gall-Stone  Disease.      1.25 
Kehr.     Gall-StoneDisease    -    2.50 
Macready  oti  Ruptures       -    6.00 
Makins.       Surgical     Experi- 
ences in  South  Africa.        -       4.00 
Maylard.     Surgery  of  the  Ali- 
mentary Canal.        -        -        3.00 
Morris.     Renal  Surgery.  2.00 

Moullin.      Complete     Text- 
book.    3d  Ed.  by  Hamilton. 
600  Illustrations.          -        -     6.00 
Smith.     Abdominal  Surg.        10.00 
Voswinkel.  Surg.  Nursing,     i.oo 
Walsham.  Surgery,  yth  Ed.  3.50 


THERAPEUTICS. 

Beasley's  3000  Prescriptions.   2.00 
Biddle.     Materia  Medica  and 

Therapeutics.     i3th  Edition. 

Coblentz.  New  Remedies,  i.oo 
Cohen.  Physiologic  Thera- 
peutics, ii  Volumes.  27.50 
Mays.  Theine.  -  -  .50 
Murray.  Notes  on  Remedies.  1.25 
Potter's  Compend.  6th  Ed.  80 

Handbook  of  Mat.  Med. 

Phar.  and  Thera.    oth  Ed.     5.00 
White   and    Wilcox.    Mat. 
Med.,  Pharmacy,  Pharmacol- 
ogy, and  Therap.     sth  Ed.     3.00 


THROAT  AND  NOSE. 

Cohen.  Throat  and  Voice.  .40 
Hall.  Nose  and  Throat.  -  2.75 
Hollopeter.  Hay  Fever.  i.oo 

Knight.  Throat.  Illus.  -  

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the  Urine.  2d  Ed.  Illus.  i.oo 
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Moullin.  The  Prostate.  •  1.75 

The  Bladder.         -          LJO 

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VENEREAL  DISEASES. 

Gowers.      Syphilis  and    the 

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Monthly  Ed.        .75  to  i.oo 


REVISED  EDITION, 


TYSON'S  PRACTICE 

A  TEXT-BOOK  FOR  PRACTITIONERS  AND  STUDENTS 
WITH  SPECIAL  REFERENCE  TO  DIAGNOSIS  AND  TREATMENT 


By  JAMES  TYSON,  M.D. 

Professor  vf  Medicine  in  the  University  of  Pennsylvania ;   Physician  to  the  University  and 
Philadelphia  Hospitals,  etc. 


COLORED  PLATES  AND  J25  OTHER  ILLUSTRATIONS 
Octavo*     J222  Pages,    Cloth,  $5.50 ;  Leather,  $6.50 ;  Half  Russia,  $7.50 


The  object  of  this  book  is — first,  to  aid  the  student  and  physician  to  recognize 
disease,  and,  second,  to  point  out  the  proper  methods  of  treatment.  To  this  end 
Diagnosis  and  Treatment  receive  special  attention,  while  pathology  and 
morbid  anatomy  have  such  consideration  as  is  demanded  by  their  importance  as  funda- 
mental conditions  of  a  thorough  understanding  of  disease.  Dr.  Tyson's  qualifications 
for  writing  such  a  work  are  unequaled.  It  is  really  the  outcome  of  over  thirty 
years'  experience  in  teaching  and  in  private  and  hospital  practice.  As  a 
teacher  he  has,  while  devoting  himself  chiefly  to  clinical  medicine,  occupied  several 
important  chairs,  notably  those  of  General  Pathology  and  Morbid  Anatomy,  and 
Clinical  Medicine  in  the  University  of  Pennsylvania,  an  experience  that  has  necessarily 
widened  his  point  of  view  and  added  weight  to  his  judgment.  This,  the  Second, 
Edition  has  been  most  thoroughly  revised,  parts  have  been  rewritten,  new 
material  and  illustrations  have  been  added,  and  in  many  respects  it  may  be  considered 
a  new  book. 

"  It  is  in  the  writing  and  preparation  of  a  work  of  this  character  that  Dr.  Tyson  stands  pre- 
eminent. Those  of  the  profession — and  there  are  many  at  this  time — who  have  been  fortunate  to 
have  been  his  pupils  during  their  medical  student  days,  will  remember  that  he  brought  to  his 
lectures  and  to  his  writings  an  amount  of  industry  and  care  which  many  other  teachers  failed  to 
bring ;  and  those  who  know  him  best  as  an  author  and  teacher  have  expected  that  his  book  on  the 
Practice  of  Medicine,  when  it  appeared,  would  be  a  credit  to  himself,  and  would  increase  his 
reputation  as  a  medical  author.  This  belief  has  proved  correct. ' ' —  Therapeutic  Gazette,  Detroit,  Mick. 

"  After  a  third  of  a  century  spent  in  the  assiduous  study,  practice,  and  teaching  of  medicine, 
and  the  publication  of  successful  books  on  various  topics,  theoretical  and  practical,  the  writing  of  a 
text-book  is  not  only  a  proper  ambition,  but  is  really  expected  by  students  and  the  profession.  So 
Professor  Tyson  best  shows  his  modesty  by  making  no  apology  for  the  present  work." — American 
Journal  of  Medical  Sciences,  Philadelphia, 

4 


j®-  Ail  prices  are  net.    No  discount  can  be  allowed  retail  purchasers. 


P.  BLAKISTON'S  SON  &  CO.'S 

Medical  and  Scientific  Publications, 


Acton.  The  Functions  and  Disorders  of  the  Reproductive  Organs 
in  Childhood,  Youth,  Adult  Age,  and  Advanced  Life,  considered  in  their  Physiological, 
Social,  and  Moral  Relations.  By  WM.  ACTON,  M.D.,  M.R.C.S.  8th  Edition.  Cloth,  $1.75 

Allen.     Commercial  Organic  Analysis. 

New  Revised  Editions.  A  Treatise  on  the  Properties,  Proximate  Analytical  Exami- 
nation and  Modes  of  Assaying  the  Various  Organic  Chemicals  and  Products  employed 
in  the  Arts,  Manufactures,  Medicine,  etc.,  with  Concise  Methods  for  the  Detection 
and  Determination  of  Impurities,  Adulterations,  and  Products  of  Decomposition,  etc. 
Revised  and  Enlarged.  By  ALFRED  H.  ALLEN,  F.C.S.,  Public  Analyst  for  the  West 
Riding  of  Yorkshire  ;  Past  President  Society  of  Public  Analysts  of  Great  Britain. 

VOL.  I.  Preliminary  Examination  of  Organic  Bodies.  Alcohols,  Neutral  Alcoholic 
Derivatives,  Ethers,  Starch  and  its  Isomers,  Sugars,  Acid  Derivatives  of  Alcohols 
and  Vegetable  Acids,  etc.  Third  Edition,  with  numerous  additions  by  the 
author,  and  revisions  and  additions  by  DR.  HENRY  LEFFMANN,  Professor  of 
Chemistry  and  Metallurgy  in  the  Pennsylvania  College  of  Dental  Surgery,  and 
in  the  Wagner  Free  Institute  of  Science,  Philadelphia,  etc.  With  many  useful 
tables.  Cloth,  $4.50 

VOL.  II — PART  I.  Fixed  Oils,  Fats,  Waxes,  Glycerin,  Soaps,  Nitroglycerin, 
Dynamite  and  Smokeless  Powders,  Wool-Fats,  Degras,  etc.  Third  Edition, 
with  many  useful  tables.  Revised  by  DR.  HENRY  LEFFMANN,  with  numerous 
additions  by  the  author.  Cloth,  $3.50 

VOL.  II — PART  II.  Hydrocarbons,  Mineral  Oils,  Lubricants,  Asphalt,  Benzene  and 
Naphthalene,  Phenols,  Creosote,  etc.  Third  Edition,  Revised  by  DR.  HENRY 
LEFFMANN,  with  additions  by  the  author.  Cloth,  $3.50 

VOL.  II — PART  III.  Terpenes,  Essential  Oils,  Resins,  Camphors,  Aromatic  Acids, 
etc.  Third  Edition.  In  Preparation. 

VOL.  Ill — PART  I.  Tannins,  Dyes,  Coloring  Matters,  and  Writing  Inks.  Third 
Edition,  Revised,  Rewritten,  and  Enlarged  by  I.  MERRITT  MATTHEWS,  Professor 
of  Chemistry  and  Dyeing  at  the  Philadelphia  Textile  School ;  Member  American 
Chemical  Society.  Cloth,  $4.50 

VOL.  Ill — PART  II.  The  Amines  and  Ammonium  Bases,  Hydrazines  and  Deriva- 
tives. Bases  from  Tar.  The  Antipyretics,  etc.  Vegetable  Alkaloids,  Tea, 
Coffee,  Cocoa,  Kola,  Cocaine,  Opium,  etc.  Second  Edition.  8vo.  Cloth,  $4. 50 

VOL.  Ill — PART  III.  Vegetable  Alkaloids  concluded,  Non-Basic  Vegetable  Bitter 
Principles.  Animal  Bases,  Animal  Acids,  Cyanogen  and  its  Derivatives,  etc. 
Second  Edition.  Cloth,  $4. 50 

VOL.  IV.  Proteids  and  Albuminous  Principles.  Proteoids  or  Albuminoids. 
Second  Edition,  with  elaborate  appendices  and  a  large  number  of  useful  tables. 

Cloth,  $4-50 
Bailey  and  Cady.     Chemical  Analysis. 

Laboratory  Guide  to  the  Study  of  Qualitative  Analysis.  By  E.  H.  S.  BAILEY,  PH.D., 
Professor  of  Chemistry,  and  HAMILTON  CADY,  A.B.,  Assistant  Professor  of  Chemistry 
in  the  University  of  Kansas.  Fourth  Edition.  Cloth,  $1.25 

8-1-02  5 


P.  BLAKISTON'S  SON  6-   CO.' S 


Ballou.     Veterinary  Anatomy  and  Physiology. 

By  WM.  R.  BALLOU,  M.D.,  late  Professor  of  Equine  Anatomy,  New  York  College  01 
Veterinary  Surgeons.  With  29  Graphic  Illustrations.  I2mo.  No.  12  fQuiz-Com- 
pendf  Series.  Cloth,  .80  ;  Interleaved  for  the  Addition  of  Notes,  $1.00 

Barrett.     Dental  Surgery 

for  General  Practitioners  and  Students  of  Medicine  and  Dentistry.  Extraction  of 
Teeth,  etc.  By  A.  W.  BARRETT,  M.D.  Third  Ed.  86  Illus.  i2mo.  Cloth,  $1.00 

Bartley.     Medical  and  Pharmaceutical  Chemistry. 

A  Text-Book  for  Medical  and  Pharmaceutical  Students.  By  E.  H.  BARTLEY,  M.D., 
Professor  of  Chemistry  and  Toxicology  at  the  Long  Island  College  Hospital ;  Dean 
and  Professor  of  Chemistry,  Brooklyn  College  of  Pharmacy  ;  Chief  Chemist,  Board 
of  Health  of  Brooklyn,  N.  Y.  Fifth  Edition,  Revised  and  Improved.  With  Illus- 
trations, Glossary,  and  Complete  Index.  i2mo.  Cloth,  $3.00;  Leather,  $3.50 

11  The  subject-matter  is  excellent.  The  descriptions  are  detailed  and  very  complete.  All  of 
these  properties  make  the  book  an  excellent  one  for  a  book  of  reference.  Indeed,  if  the  book  be 
considered  in  the  light  of  this  purpose,  it  is  hard  to  find  anything  in  it  for  adverse  criticism." — 
Boston  Medical  and  Surgical  Journal. 

Clinical  Chemistry. 

The  Chemical  Examination  of  the  Saliva,  Gastric  Juice,  Feces,  Milk,  Urine,  etc., 
with  Notes  on  Urinary  Diagnosis,  Volumetric  Analysis,  and  Weights  and  Meas- 
ures. Illustrated.  I2mo.  Cloth,  $1.00 

Beale.     On  Slight  Ailments. 

Their  Nature  and  Treatment.  By  LIONEL  S.  BEALE,  M.D.,  F.R.S.,  Professor  of 
Practice,  King's  Medical  College,  London.  Second  Edition.  8vo.  Cloth,  $1.25 

Beasley's  Book  of  Prescriptions. 

Containing  over  3100  Prescriptions,  collected  from  the  Practice  of  the  most  Eminent 
Physicians  and  Surgeons — English,  French,  and  American  ;  a  Compendious  History 
of  the  Materia  Medica,  Lists  of  the  Doses  of  all  Officinal  and  Established  Prepa- 
rations, and  an  Index  of  Diseases  and  their  Remedies.  By  HENRY  BEASLEY. 
Seventh  Edition.  Cloth,  $2.00 

Druggists'  General  Receipt  Book. 

Comprising  a  copious  Veterinary  Formulary  ;  Recipes  in  Patent  and  Proprietary 
Medicines,  Druggists'  Nostrums,  etc. ;  Perfumery  and  Cosmetics  ;  Beverages, 
Dietetic  Articles,  and  Condiments  ;  Trade  Chemicals,  Scientific  Processes,  and 
an  Appendix  of  Useful  Tables.  Tenth  Edition.  Cloth,  $2.00 

Pharmaceutical  Formulary 

and  Synopsis  of  the  British,  French,  German,  and  United  States  Pharmacopoeias. 
Comprising  Standard  and  Approved  Formulae  for  the  Preparations  and  Com- 
pounds Employed  in  Medical  Practice.  Twelfth  Edition.  Cloth,  $2.00 

Berry.     The  Thyroid  Gland. 

The  Diseases  of  the  Thyroid  Gland  and  Their  Surgical  Treatment.  By  JAMES 
BERRY, 'M.B.,  B.S.,  F.R.C.S.,  Surgeon  to  the  Royal  Free  Hospital.  121  Illustrations, 
from  Original  Photographs  of  Cases.  Cloth,  $4.00 

Biddle's  Materia  Medica  and  Therapeutics.     Thirteenth  Edition. 
Including  Dose  List,  Dietary  for  the  Sick,  Table  of  Parasites,  and  Memoranda  of 
New  Remedies.     By  the  late  JOHN  B.  BIDDLE,  M.D.     Thirteenth  Edition,  Revised 
by  CLEMENT   BIDDLE,  M.D.,  Assistant  Surgeon  U.  S.  Navy.     With  64  Illustrations 
and  a  Clinical  Index.     Octavo. 


MEDICAL  AND   SCIENTIFIC  PUBLICATIONS. 


Bigelow.     Plain  Talks  on  Medical  Electricity  and  Batteries. 
With  a  Therapeutic  Index  and  a  Glossary.     By  HORATIO  R.  BIGELOW,  M.D.     With 
43  Illustrations  and  a  Glossary.     Second  Edition.  Cloth,  $1.00 

Birch.      Practical  Physiology. 

An  Elementary  Class-Book.  Including  Histology,  Chemical  and  Experimental 
Physiology.  By  DE  BURGH  BIRCH,  M.D.,  C.M.,  F.R.S.E.,  Professor  of  Physiology  in 
the  Yorkshire  College  of  the  Victoria  University,  etc.  62  Illus.  I2mo.  Cloth,  $1.75 

Bishop.     Uterine  Fibromyomata. 

Their  Pathology,  Diagnosis,  and  Treatment.  By  E.  STANMORE  BISHOP,  F.R.C.S. 
(Eng.),  President  Manchester  Clinical  Society  ;  Fellow  British  Gynecological  Society. 
Full-page  Plates  and  other  Illustrations.  Octavo.  Cloth,  $3.50 

Black.      Micro-Organisms. 

The  Formation  of  Poisons.  A  Biological  Study  of  the  Germ  Theory  of  Disease. 
By  G.  V.  Black,  M.D.,  D.D.S.  Cloth,  .75 

Blackburn.     Autopsies.     Illustrated. 

A  Manual  of  Autopsies,  Designed  for  the  Use  of  Hospitals  for  the  Insane  and  other 
Public  Institutions.  By  I.  W.  BLACKBURN,  M.D.,  Pathologist  to  the  Government 
Hospital  for  the  Insane.  Illustrated.  Cloth,  $1.25 

Bloxam.     Chemistry  (Inorganic  and  Organic). 

With  Experiments.  By  CHARLES  L.  BLOXAM.  Edited  by  J.  M.  THOMPSON,  Pro- 
fessor of  Chemistry  in  King's  College,  London,  and  A.  G.  BLOXAM,  Head  of  the 
Chemistry  Department,  Goldsmith's -Institute,  London.  Ninth  Edition,  Revised  and 
Enlarged.  281  Engravings.  8vo.  Preparing. 

Bracken.     Outlines  of  Materia  Medica  and  Pharmacology. 

By  H.  M.  BRACKEN,  Professor  of  Materia  Medica  and  Therapeutics  and  of  Clinical 
Medicine,  University  of  Minnesota.  8vo.  Cloth,  $2.75 

Broomell.     Anatomy  and  Histology  of  the  Mouth  and  Teeth. 
By  DR.  I.  N.  BROOMELL,  Professor  of  Dental  Anatomy,  Dental  Histology,  and  Pros- 
thetic Technics  in  the  Pennsylvania  College  of  Dental  Surgery.     Second  Edition, 
Enlarged  and  Revised.     337    handsome   Illustrations,   the   majority  of   which  are 
original.     Large  Octavo.     Just  Ready.  Cloth,  $4. 50 

Brown.     Medical  Diagnosis.     Fourth  Edition. 

A  Manual  of  Clinical  Methods.  By  J.  J.  GRAHAM  BROWN,  M.D.,  F.R.C.P.,  Lecturer 
on  Principles  and  Practice  of  Medicine  in  the  School  of  Medicine  of  the  Royal 
Colleges,  Edinburgh.  Fourth  Edition.  112  Illustrations.  I2mo.  Cloth,  $2.25 

Brubaker.     Compend  of  Physiology.     Tenth  Edition. 

A  Compend  of  Physiology,  specially  adapted  for  the  use  of  Students  and  Physicians. 
By  A.  P.  BRUBAKER,  M.D.,  Adjunct  Professor  of  Physiology  at  Jefferson  Medical 
College  ;  Professor  of  Physiology,  Pennsylvania  College  of  Dental  Surgery,  Philadel- 
phia. Tenth  Edition,  Revised,  Enlarged,  and  Illustrated.  No.  4  f  Quiz-  Compend  f 
Series.  I2mo.  Cloth,  .80;  Interleaved,  $1.00 

Bulkley.     The  Skin  in  Health  and  Disease. 
By  L.  DUNCAN  BULKLEY,  M.D.     Illustrated.  Cloth.  .40 

Bunge.     Physiologic  and  Pathologic  Chemistry. 

By  DR.  C.  BUNGE,  Professor  at  Basel.  Authorized  Translation  from  the  Fourth 
German  Edition.  Edited  by  E.  A.  STARLING,  M.D.,  F.R.S.,  Professor  of  Physiology 
in  University  College,  London.  Octavo.  Cloth,  $3.00 


P.  BLAKISTON'S  SON  6-   CO.' S 


Burnet.     Foods  and  Dietaries. 

A  Manual  of  Clinical  Dietetics.  By  R.  W.  BURNET,  M.D.,  M.R.C.P.,  Physician  to 
the  Great  Northern  Central  Hospital.  With  Appendix  on  Predigested  Foods  and 
Invalid  Cookery.  Full  Directions  as  to  Hours  of  Taking  Nourishment,  Quantity, 
etc.  Third  Edition.  Cloth,  $1.50 

Burnett.     Hearing  and  How  to  Keep  It. 

By  CHAS.  H.  BURNETT,  M.D.,  Professor  of  Diseases  of  the  Ear  at  the  Philadelphia 
Polyclinic.  Illustrated.  Cloth,  .40 

Butlin.  The  Operative  Surgery  of  Malignant  Disease. 
By  HENRY  T.  BUTLIN,  F.R.C.S.,  Assistant  Surgeon  to,  and  Demonstrator  of  Surgery 
at,  St.  Bartholomew's  Hospital,  London,  etc.,  assisted  by  JAMES  BERRY,  F.R,C.S., 
WM.  BRUCE-CLARKE,  M.B.,  F.R.C.S.,  A.  H.  G.  DORAN,  F.R.C.S.,  PERCY  FURNIVALL, 
F.R.C.S.,  W.  H.  H.  JESSOP,  M.B.,  F.R.C.S.,  and  H.  J.  WARING,  B.SC.,  F.R.C.S. 
Second  Edition,  Revised  and  Rewritten.  Illustrated.  Octavo.  Cloth,  #4.50 

Buxton.     On  Anesthetics. 

A  Manual.  By  DUDLEY  WILMOT  BUXTON,  M.R.C.S.,  M.R.C.P.,  Assistant  to  Professor 
of  Medicine  and  Administrator  of  Anesthetics,  University  College  Hospital,  London. 
Third  Edition,  Illustrated.  I2mo.  Cloth,  $1.50 

Byford.     Manual  of  Gynecology.     363  Illustrations. 

By  HENRY  T.  BYFORD,  M.D.,  Professor  of  Gynecology  and  Clinical  Gynecology  in 
the  College  of  Physicians  and  Surgeons  of  Chicago,  and  in  Post-Graduate  Medical 
School,  etc.  Third  Edition,  Revised  and  Enlarged.  363  Illustrations,  many  of  which 
are  from  original  drawings  and  several  of  which  are  Colored.  I2mo.  Just  Ready. 

Cloth,  $3.00 

Caldwell.     Chemical  Analysis. 

Elements  of  Qualitative  and  Quantitative  Chemical  Analysis.  By  G.  C.  CALDWELL, 
B.S.,  PH.D.,  Professor  of  Agricultural  and  Analytical  Chemistry  in  Cornell  Univer- 
sity, Ithaca,  New  York,  etc.  Third  Edition.  Octavo.  Cloth,  $1.00 

Cameron.     Oils  and  Varnishes. 

By  JAMES  CAMERON,  F.I. c.    Illustrations,  Formulae,  Tables,  etc.     i2mo.     Cloth,  $2.25 

Soap  and  Candles. 

A  Handbook  for  Manufacturers,  Chemists,  etc.  54  Illustrations.    I2mo.  Cloth,  $2.00 
Campbell.     Dissection  Outline  and  Index. 

A  Systematic  Outline  for  Students  for  the  Dissection  of  the  Human  Body  and  an 
Arranged  Index  adapted  for  Use  with  Morris'  Anatomy.  By  WILLIAM  A.  CAMPBELL, 
B.S.,  M.D.,  formerly  Demonstrator  of  Anatomy  in  the  Medical  Department  of  the 
University  of  Michigan,  Ann  Arbor.  Second  Edition,  Revised.  Cloth,  .  50 

Canfield.     Hygiene  of  the  Sick-Room. 

Being  a  Brief  Consideration  of  Asepsis,  Antisepsis,  Disinfection,  Bacteriology,  Immu- 
nity, Heating  and  Ventilation,  and  kindred  subjects,  for  the  Use  of  Nurses  and  other 
Intelligent  Women.  By  WILLIAM  BUCKINGHAM  CANFIELD,  A.M.,  M.D.,  late  Lecturer 
on  Clinical  Medicine,  University  of  Maryland.  I2mo.  Cloth,  $1.25 

Carpenter.     The  Microscope  and  Its  Revelations. 
By  W.  B.   CARPENTER,   M.D.,   F.R.S.     Eighth  Edition,  by  REV.   DR.   DALLINGER, 
F.R.S.     Rewritten,  Revised,  and  Enlarged.     817  Illustrations  and  23  Plates.     Octavo. 
1 1 36  pages.     Just  Ready.  Cloth,  $8.00;  Half  Morocco,  $9.00 

Chase.     General  Paresis. 

By  ROBERT  H.  CHASE,  M.D.,  Physician-in-chief  Friends'  Asylum  for  the  Insane,  Frank- 
ford,  Philadelphia  ;  late  Resident  Physician  State  Hospital  for  Insane,  Norristown, 
Pennsylvania,  etc.  Illustrated.  Just  Ready.  Cloth,  $1.75 


MEDICAL  AND   SCIENTIFIC  PUBLICATIONS.  9 

Cazeaux  and  Tarnier's  Midwifery.  With  Appendix,  by  Munde. 
The  Theory  and  Practice  of  Obstetrics,  including  the  Diseases  of  Pregnancy  and 
Parturition,  Obstetrical  Operations,  etc.  By  P.  CAZEAUX.  Remodeled,  rearranged, 
and  revised  by  S.  TARNIER,  M.D.  Eighth  American  from  the  Eighth  French  and 
First  Italian  Edition.  Edited  by  ROBERT  J.  HESS,  M.D.  With  an  Appendix  by  PAUL 
F.  MUNDE,  M.D.  Illustrated  by  Lithographs,  Full-page  Plates,  and  numerous  En- 
gravings. 8vo.  Cloth,  $4.50;  Full  Leather,  $5.50 

Clowes  and  Coleman.     Quantitative  Analysis. 

Adapted  for  the  Use  of  the  Laboratories  of  Schools  and  Colleges.  By  FRANK  CLOWES, 
SC.D.,  Emeritus  Professor  of  Chemistry,  University  College,  Nottingham,  and  I. 
BERNARD  COLEMAN,  Assoc.  R.  C.  Sci.,  Dublin,  Professor  of  Chemistry,  Southwest 
London  Polytechnic.  Fifth  Edition.  122  Illustrations.  Cloth,  $3.50 

Coblentz.      Manual  of  Pharmacy. 

A  Text-Book  for  Students.  By  VIRGIL  COBLENTZ,  A.M.,  PH.D:,  F.C.S.,  Professor  of 
Chemistry  and  Physics  ;  Director  of  Pharmaceutical  Laboratory,  College  of  Phar- 
macy of  the  City  of  New  York.  Second  Edition,  Revised  and  Enlarged.  437  Illus- 
trations. Octavo.  572  pages.  Cloth,  $3.50  ;  Sheep,  $4.50 

The  Newer  Remedies. 

Including  their  Synonyms,  Sources,  Methods  of  Preparation,  Tests,  Solubilities, 
and  Doses  as  far  as  known.  Together  with  Sections  on  Organo-Therapeutic 
Agents  and  Indifferent  Compounds  of  Iron.  Third  Edition,  very  much  enlarged. 
Octavo.  Cloth,  $1.00 

Volumetric  Analysis. 

A  Practical  Handbook  for  Students  of  Chemistry.  Including  Indicators,  Test- 
Papers,  Alkalimetry,  Acidimetry,  Analysis  by  Oxidation  and  Reduction,  lodom- 
etry,  Assay  Processes  for  Drugs  with  the  Titrimetric  Estimation  of  Alkaloids, 
Estimation  of  Phenol,  Sugar,  Tables  of  Atomic  and  Molecular  Weights. 
Illustrated.  8vo.  Cloth,  $1.25 

Cohen.     System  of  Physiologic  Therapeutics.      Illustrated. 

A  Practical  Exposition  of  the  Methods  Other  than  Drug-giving,  Useful  in  the  Treat- 
ment of  the  Sick  and  in  the  Prevention  of  Disease.  Edited  by  SOLOMON  SOLIS 
COHEN,  A.M.,  M.D.,  Professor  of  Medicine  and  Therapeutics  in  the  Philadelphia 
Polyclinic  ;  Lecturer  on  Clinical  Medicine  at  Jefferson  Medical  College  ;  Physician 
to  the  Philadelphia  Hospital  and  to  the  Rush  Hospital  for  Consumption  ;  formerly 
Lecturer  on  Therapeutics,  Dartmouth  Medical  College.  To  be  issued  in  Eleven 
Compact  Octavo  Volumes.  Complete  Set,  Cloth,  $27.50;  Half  Morocco,  $38.50 

Electrotherapy.     220  Illustrations.     Two  Volumes.     Ready. 

By  GEORGE  W.  JACOBY,  M.D.,  New  York,  Consulting  Neurologist  to  the  German 
Hospital,  to  the  Infirmary  for  Women  and  Children,  to  the  Craig  Colony  for 
Epileptics,  etc.  Special  articles  by  EDWARD  JACKSON,  A.M.,  M.D.,  Denver, 
Col. ;  Emeritus  Professor  of  Diseases  of  the  Eye  in  the  Philadelphia  Polyclinic  ; 
Member  American  Ophthalmological  Society  ;  Fellow  and  ex-President  American 
Academy  of  Medicine,  etc. — By  WILLIAM  SCHEPPEGRELL,  M.D.,  New  Orleans, 
ex-Vice-President  American  Laryngological,  Rhinological,  and  Otological 
Society. — By  J.  CHALMERS  DA  COSTA,  M.D.,  Clinical  Professor  of  Surgery  in 
Jefferson  Medical  College;  Surgeon  to  the  Philadelphia  and  to  St.  Joseph's 
Hospitals,  etc. — By  FRANKLIN  H.  MARTIN,  M.D.,  Professor  of  Gynecology,  Post- 
Graduate  Medical*  School  of  Chicago  ;  Gynecologist  Chicago  Charity  Hospital ; 
Chairman  Section  of  Obstetrics  and  Diseases  of  Women  of  the  American  Medi- 
cal Association  (1895),  etc. — By  A.  H.  OHMANN-DUMESNIL,  M.D.,  Editor  St. 
Louis  Medical  and  Surgical  Journal ;  Member  International  Dermatological 
Congress  ;  formerly  Professor  of  Dermatology,  St.  Louis  Medical  College,  etc. 


10  P.   BLAKISTON'S  SON  &*    CO.' S 

Cohen.     Physiologic  Therapeutics. — Continued. 

Climatology   and   Health    Resorts,    Including   Mineral    Springs. 
Two  Volumes,  with  Colored  Maps.     Ready. 

By  F.  PARKES  WEBER,  M.A.,  M.D.,  F.R.C.P.  (Lond.),  Physician  to  the  German 
Hospital,  Dalston  ;  Assistant  Physician  North  London  Hospital  for  Consump- 
tion ;  Author  of  "  The  Mineral  Waters  and  Health  Resorts  of  Europe;"  and 
GUY  HINSDALE,  A.M.,  M.D.,  Secretary  of  the  American  Climatological  Associa- 
tion ;  President  of  the  Pennsylvania  Society  for  the  Prevention  of  Tuberculosis, 
etc.  Including  an  article  on  Hawaii  by  TITUS  MUNSON  COAN,  M.D.,  of  New 
York.  With  Colored  Maps,  prepared  by  DR.  W.  F.  R.  PHILLIPS,  of  the  U.  S. 
Weather  Bureau,  Washington,  D.  C. 

Prophylaxis — Personal  Hygiene — Care  of  the  Sick.     Illustrated. 

By  DR.  JOSEPH  MCFARLAND,  Professor  of  Pathology,  Medico-Chirurgical  College, 
Philadelphia;  DR.  HENRY  LEFFMANN,  Professor  of  Chemistry  in  the  Woman's 
Medical  College,  Philadelphia;  ALBERT  ABRAMS,  A.M.,  M.D.  (University  of 
Heidelberg),  formerly  Professor  of  Pathology,  Cooper  Medical  College,  San 
Francisco  ;  and  DR.  W.  WAYNE  BABCOCK,  Lecturer  on  Pathology  and  Bac- 
teriology, Medico-Chirurgical  College,  Philadelphia. 

Dietotherapy  :  Food  in  Health  and  Disease.     Ready. 

By  NATHAN  S.  DAVIS,  JR.,  A.M.,  M.D.,  Professor  of  Principles  and  Practice  of 
Medicine  in  Northwestern  University  Medical  School ;  Physician  to  Mercy  Hos- 
pital, Chicago  ;  Member  American  Academy  of  Medicine,  American  Climato- 
logical Society,  etc.  With  Tables  of  Dietaries,  Relative  Value  of  Foods,  etc. 

Mechanotherapy  and  Physical  Education.     Illustrated. 

By  JOHN  KEARSLEY  MITCHELL,  M.D.,  Assistant  Physician  to  the  Orthopedic 
Hospital  and  Infirmary  for  Nervous  Diseases  ;  Assistant  Neurologist  Presbyterian 
Hospital,  Philadelphia,  etc. ;  formerly  Lecturer  on  Symptomology  at  the  Univer- 
sity of  Pennsylvania;  and  LUTHER  GULICK,  M.D.,  of  Brooklyn,  N.  Y.,  formerly 
of  Springfield,  Mass.,  Mem.  American  Association  for  Advancement  of  Physical 
Education,  Amer.  Medical  Association,  etc.  With  a  Chapter  on  Orthopedic 
Appliances  by  JAMES  K.  YOUNG,  M.D.,  Professor  of  Orthopedic  Surgery,  Phila- 
delphia Polyclinic  ;  Instructor  in  Orthopedic  Surgery,  University  of  Pennsylvania  ; 
and  an  Article  on  Ocular  Orthopedics  by  WALTER  L.  PYLE,  M.D. 

Rest — Mental  Therapeutics — Suggestion. 

By  FRANCIS  X.  DERCUM,  M.D.,  Clinical  Professor  of  Nervous  Diseases  in  Jeffer- 
son Medical  College  ;  Neurologist  to  the  Philadelphia  Hospital ;  Consulting 
Physician  to  the  State  Asylum  for  the  Chronic  Insane  at  Wernersville,  Penna. ; 
Consulting  Neurologist  to  St.  Agnes'  Hospital ;  Neurologist  to  the  Jewish  Hos- 
pital of  Philadelphia. 

Hydrotherapy  —  Thermotherapy  —  Heliotherapy —  Crounother- 

apy — Phototherapy — Balneology.     Ready. 

By  DR.  WILHELM  WINTERNITZ,  Professor  of  Clinical  Medicine  in  the  University 
of  Vienna  ;  Director  of  the  General  Polyclinic  in  Vienna,  etc. ;  assisted  by 
DR.  ALOIS  STRASSER,  Instructor  in  Clinical  Medicine,  University  of  Vienna  ; 
and  DR.  B.  BUXBAUM,  Chief  Physician  of  the  Hydrotherapeutic  Institute  of 
Vienna  ;  and  DR.  E.  HEINRICH  KISCH,  Professor  in  the  University  of  Prague  ; 
Physician  at  Marienbad  Spa.  With  Special  Chapters  by  DR.  A.  C.  PEALE,  of  the 
National  Museum,  Washington,  D.  C.,  DR.  J.  H.  KELLOGG,  Battle  Creek, 
Mich.,  and  HARVEY  GUSHING,  M.D.,  Johns  Hopkins  Hospital,  Baltimore,  and 
an  Appendix  by  DR.  COHEN. 

Pneumatotherapy  and  Inhalation  Methods.     Illustrated. 
By  DR.  PAUL  TISSIER,  Chief  of  Clinic  of  the  Faculty  of  Medicine  of  Paris. 


MEDICAL  AND   SCIENTIFIC  PUBLICATIONS.  11 

Cohen.     Physiologic  Therapeutics. — Continued. 

Serotherapy — Organotherapy — Blood-Letting,  etc. — Principles  of 
Therapeutics — Digest — I  ndex. 

By  JOSEPH  MCFARLAND,  M.D.,  Professor  of  Pathology  in  the  Medico-Chirurgical 
College,  Philadelphia  ;  Pathologist  to  the  Medico  Chirurgical  Hospital,  etc. — 
O.  T.  OSBORNE,  M.D.,  Professor  of  Materia  Medica  and  Therapeutics,  Medical 
Department,  Yale  University,  New  Haven. — FREDERICK  A.  PACKARD,  M.D., 
Visiting  Physician  to  the  Pennsylvania  and  to  the  Children's  Hospitals. — The 
Editor,  and  AUGUSTUS  A.  ESHNER,  M.D.,  Professor  of  Clinical  Medicine  in  the 
Philadelphia  Polyclinic  ;  Physician  to  the  Philadelphia  Hospital,  etc. 

*#*  Complete  descriptive  circular  upon  application. 

"  There  is  surely  room  for  just  such  a  set  of  books.  We  have  been  too  prone  to  think  that  we 
were  teaching  therapeutics  sufficiently  when  we  taught  our  students  the  old  materia  medica  and  the 
use  of  mere  drugs,  forgetful  and  careless  of  the  importance  of  the  therapeutic  value  of  the  meihods 
of  which  this  series  of  books  will  speak." — Johns  Hopkins  Hospital  Bulletin. 

Cohen.     The  Throat  and  Voice. 

By  J.  SOLIS  COHEN,  M.D.     Illustrated.      I2mo.  Cloth,  .40 

Congdon.     Laboratory  Instructions  in  General  Chemistry. 

By  ERNEST  A.  CONGDON,  Professor  of  Chemistry  in  the  Drexel  Institute,  Philadelphia  ; 
Member  American  Chemical  Society  ;  Fellow  of  the  London  Chemical  Society,  etc. 
With  an  Appendix,  useful  Tables,  and  56  Illustrations.  Interleaved,  Cloth,  $1.00 

Conn.     Agricultural  Bacteriology. 

Including  a  Study  of  Bacteria  as  Relating  to  Agriculture,  with  Special  Reference  to 
the  Bacteria  in  Soil,  in  the  Dairy,  in  Food  Products,  in  Domestic  Animals,  and  in 
Sewage.  By  H.  W.  CONN,  PH.D.,  Professor  of  Biology,  Wesleyan  University, 
Middletown,  Conn.;  Author  of  "Evolution  of  To-day,"  "  The  Story  of  Germ  Life," 
etc.  With  Illustrations.  Cloth,  $2.50 

The  Relation  of  Bacteria  to  Milk  Products. 

Designed  for  Students  of  Dairying,  Boards  of  Health,  and  Bacteriologists.  Illus- 
trated. I2mo.  In  Press. 

Coplin.     Manual  of  Pathology.     Third  Edition.     330  Illustrations. 

Including  Bacteriology,  the  Technic  of  Post-mortems,  and  Methods  of  Pathologic 
Research.  By  W.  M.  LATE  COPLIN,  M.D.,  Professor  of  Pathology  and  Bacteriology, 
Jefferson  Medical  College  ;  Pathologist  to  Jefferson  Medical  College  Hospital  and  to 
the  Philadelphia  Hospital  ;  Bacteriologist  to  the  Pennsylvania  State  Board  of  Health. 
Third  Edition,  Rewritten  and  Enlarged.  330  Illustrations,  many  of  which  are  origi- 
nal, and  7  Colored  Plates.  8vo.  Cloth,  $3.50 

Practical  Hygiene. 

With  Special  Articles  on  Plumbing,  Ventilation,  etc.  138  Illustrations.  8vo. 
Second  Edition.  In  Preparation. 

Crocker.     Diseases  of  the  Skin.     Third  Edition. 

Their  Description,  Pathology,  Diagnosis,  and  Treatment,  with  Special  Reference  to 
the  Skin  Eruptions  of  Children.  By  H.  RADCLIFFE  CROCKER,  M.D.,  Physician  to 
the  Department  of  Skin  Diseases,  University  College  Hospital,  London.  Third  Edi- 
tion, Thoroughly  Revised,  with  new  Illustrations.  Nearly  Ready.  Cloth,  $5.00 

Cuff.     Lectures  on  Medicine  to  Nurses. 

By  HERBERT  EDMUND  CUFF,  M.D.,  late  Assistant  Medical  Officer,  Stockwell  Fever 
Hospital,  England.  Third  Edition,  Revised.  With  25  Illustrations.  Cloth,  $1.25 


12  P.  BLAKISTON'S  SON  6-   CO:  S 

Gushing.     Compend  of  Histology. 

Specially  adapted  for  the  use  of  Medical  Students  and  Physicians.  By  H.  H.  GUSHING, 
M.D.,  Director  of  Histological  and  Embryological  Laboratories,  Woman's  Medical 
College  of  Pennsylvania  ;  Demonstrator  of  Histology  and  Embryology,  Jefferson 
Medical  College,  Philadelphia.  Illustrated.  No.  17  ?  Quiz- Compend?  Series. 
I2mo.  In  Press.  Cloth,  .80;  Interleaved  for  Notes,  $1.00 

Davis.     Dietotherapy.     Food  in  Health  and  Disease. 
See  COHEN,  Physiologic  Therapeutics,  page  10. 

Davis.     Essentials  of  Materia  Medica  and  Prescription  Writing. 
By  J.  AUBREY  DAVIS,  M.D.     izmo.  Cloth,  $1.50 

Davis.     The  Principles  and  Practice  of  Bandaging. 

By  GWILYM  G.  DAVIS,  M.D.,  M.R.C.S.,  Universities  of  Pennsylvania  and  Gottingen, 
Assistant  Demonstrator  of  Surgery,  University  of  Pennsylvania  ;  Surgeon  to  the  Out- 
Patient  Departments  of  the  Episcopal  and  Children's  Hospitals  ;  Assistant  Surgeon 
to  the  Orthopaedic  Hospital.  Second  Edition,  Revised  and  Rewritten.  163  Illustra- 
tions, Redrawn  specially  for  this  edition.  Just  Ready.  Cloth,  $1.50 

Domville.     Manual  for  Nurses 

and  Others  Engaged  in  Attending  to  the  Sick.  By  ED.  J.  DOMVILLE,  M.D.  Ninth 
Edition,  Revised.  With  Recipes  for  Sick-room  Cookery,  etc.  I2mo.  In  Press. 

Donders.     Refraction.     Portrait  of  Author. 

An  Essay  on  the  Nature  and  the  Consequences  of  Anomalies  of  Refraction.  By  F. 
C.  DONDERS,  M.D.  Authorized  Translation.  Revised  and  Edited  by  CHARLES  A. 
OLIVER,  A.M.,  M.D.  (Univ.  Pa.),  one  of  the  Attending  Surgeons  to  the  Wills  Eye 
Hospital.  With  a  Portrait  of  the  Author.  Octavo.  Half  Morocco,  Gilt,  $1.25 

Da  Costa.     Clinical  Hematology.     Colored  Plates. 

A  Practical  Guide  to  the  Examination  of  the  Blood  by  Clinical  Methods,  with  Refer- 
ence to  the  Diagnosis  of  Disease.  By  JOHN  C.  DA  COSTA,  JR.,  M.D.,  Assistant 
Demonstrator  of  Clinical  Medicine  in  the  Jefferson  Medical  College,  Philadelphia  ; 
Assistant  in  the  Medical  Clinic,  Jefferson  Medical  College  Hospital ;  Haematologist 
to  the  German  Hospital.  With  six  Colored  Plates  and  48  other  Illustrations.  Octavo. 
Just  Ready.  Cloth,  $5.00;  Sheep,  $6.00 

Deaver.  Surgical  Anatomy.  450  Full-page  Plates. 
A  Treatise  on  Human  Anatomy  in  its  Application  to  the  Practice  of  Medicine  and 
Surgery.  By  JOHN  B.  DEAVER,  M.D.,  Surgeon-in-Chief  to  the  German  Hospital  ; 
Surgeon  to  the  Children's  Hospital  and  to  the  Philadelphia  Hospital;  Consulting 
Surgeon  to  St.  Agnes',  St.  Timothy's,  and  Germantown  Hospitals  ;  formerly  Assistant 
Professor  of  Applied  Anatomy,  University  of  Pennsylvania,  etc.  With  over  450 
very  handsome  Full-page  Illustrations  engraved  from  original  drawings  made  by 
special  artists  from  dissections  prepared  for  the  purpose  in  the  dissecting-rooms  of  the 
University  of  Pennsylvania.  Three  large  volumes.  Royal  square  octavo.  Sold  by 
Subscription.  Orders  taken  for  complete  sets  only.  Description  upon  Application. 
Cloth,  $21.00  ;  Half  Morocco  or  Sheep,  $24.00  ;  Half  Russia,  $27.00 

SYNOPSIS  OF  CONTENTS. 
VOLUME  I. — Upper  Extremity — Back  of  Neck,  Shoulder,  and  Trunk — Cranium 

— Scalp — Face . 
VOLUME  II. — Neck — Mouth,   Pharynx,   Larynx,   Nose — Orbit — Eyeball — Organ 

of  Hearing — Brain — Female  Perineum — Male  Perineum. 

VOLUME  III. — Abdominal  Wall — Abdominal  Cavity — Pelvic    Cavity — Chest — 
Lower  Extremity. 

See  next  page  for  Reviews. 


MEDICAL  AND   SCIENTIFIC  PUBLICATIONS.  13 

DEAVER'S  SURGICAL  ANATOMY 


The  illustrations,  which  at  the  first  glance  appear  as  the  prominent  feature  of 
the  book- -but  which  in  reality  do  not  overshadow  the  text — consist  of  a  series  of 
pictures  absolutely  unique  and  fresh.  They  will  bear  comparison  from  an  artistic  point 
of  view  with  any  other  work,  while  from  a  practical  point  of  view  there  is  no  other 
volume  or  series  of  volumes  to  which  they  can  be  compared.  When  originally  an- 
nounced, the  book  was  to  contain  two  hundred  illustrations.  As  the  work  of  prepara- 
tion progressed,  this  number  gradually  increased  to  more  than  four  hundred  and  fifty 
full-page  plates,  many  of  which  contain  more  than  one  figure.  With  the  exception  of 
a  few  minor  pictures  made  from  preparations  in  the  possession  of  the  author,  they  have 
all  been  drawn  by  special  artists  from  dissections  made  for  the  purpose  in  the  dissecting- 
rooms  of  the  University  of  Pennsylvania.  Their  accuracy  cannot  be  questioned,  as 
each  drawing  has  been  submitted  to  the  most  careful  scrutiny. 

From  The  Medical  Record,  New  York. 

44  The  reader  is  not  only  taken  by  easy  and  natural  stages  from  the  more  superficial  to  the 
deeper  regions,  but  the  various  important  regional  landmarks  are  also  indicated  by  schematic 
tracing  upon  the  limbs.  Thus  the  courses  of  arteries,  veins,  and  nerves  are  indicated  in  a  way  that 
makes  the  lesson  strikingly  impressive  and  easily  learned.  No  expense,  evidently,  has  been 
spared  in  the  preparation  of  the  work,  judging  from  the  number  of  full-page  plates  it  contains,  not 
counting  the  smaller  drawings.  Most  of  these  have  been  '  drawn  bv  .special  artists  from  dissections 
made  for  the  purpose  in  the  dissecting-rooms  of  the  University  of  Pennsylvania.'  In  summing  up 
the  general  excellences  of  this  remarkable  work,  we  can  accord  our  unqualified  praise  for  the 
accurate,  exhaustive,  and  systematic  manner  in  which  the  author  has  carried  out  his  plan,  and  we 
can  commend  it  as  a  model  of  its  kind,  which  must  be  possessed  to  be  appreciated/' 

From  The  Philadelphia  Medical  Journal. 

"  Many  members  of  the  profession  to  whom  Dr.  Deaver  is  well  known  either  personally  or  by 
reputation  as  a  surgeon,  writer,  teacher,  and  practical  anatomist,  have  awaited  the  appearance  of 
his  Surgical  Anatomy  with  the  expectation  of  finding  in  it  a  guide  in  this  difficult  branch  of  medi- 
cine of  much  more  than  ordinary  practical  value,  and  their  expectations  will  not  be  disappointed." 

From  The  Journal  of  the  American  Medical  Association. 

"  In  order  to  show  its  thoroughness,  it  is  only  necessary  to  mention  that  no  less  than  twelve 
full-page  plates  are  reproduced  in  order  to  accurately  portray  the  surgical  anatomy  of  the  hand, 
and  it  is  doubtful  whether  any  better  description  exists  in  any  work  in  the  English  language/' 

From  The  Southern  California  Practitioner. 

"  Aside  from  the  merit  of  this  great  work,  it  will  be  a  delight  to  the  lover  of  books.  Its  gen. 
eral  make-up  shows  the  highest  development  of  the  book-making  art.  The  bibliophile,  when 
holding  one  of  these  volumes  in  his  hands,  would  be  as  careful  with  it  as  though  he  were  handling 
an  infant,  and  to  drop  it  would  cause  him  the  keenest  pain.  The  illustrations,  the  print,  and  the 
paper  and  binding  are  each  and  all  delightful  in  themselves,  and  yet  the  text  is  concise  and  clear, 
and  taken  with  the  illustrations  make  a  remarkably  good  substitute  for  the  dissecting-room.  To 
have  these  three  volumes  on  his  library  shelves  will  be  a  source  of  pride  and  joy  and  profit  to 
every  practitioner.  Dr.  Deaver  has  in  these  volumes  conferred  a  boon  upon  the  medical  profession 
which  has,  at  least,  never  been  surpassed  by  any  one." 

From  The  New  Orleans  Medical  and  Surgical  Journal. 

"  While  the  needs  of  the  undergraduate  have  been  fully  kept  in  view,  it  has  been  the  aim  of 
the  author  to  provide  a  work  which  would  be  sufficient  for  reference  for  use  in  actual  practice.  We 
believe  the  book  fulfils  both  requirements.  The  arrangement  is  systematic  and  the  discussion  of 
surgical  relations  thorough ." 

Large  Descriptive  Circular  will  be  sent  upon  application 


14  P.   BLAKISTON'S  SON  6-    CO.'S 

Deaver.     Appendicitis.     Third  Edition. 

Its  History,  Anatomy,  Etiology,  Pathology,  Symptoms,  Diagnosis,  Prognosis,  Treat- 
ment, Complications,  and  Sequelae.  With  22  Plates,  10  of  which  are  Colored. 
Third  Edition,  Revised  and  Rewritten.  Preparing. 

Dercum.     Rest — Mental  Therapeutics — Suggestion. 
See  COHEN,  Physiologic  Therapeutics,  page  10. 

Diihrssen.     A  Manual  of  Gynecological  Practice. 

By  DR.  A.  DUHRSSEN,  Privat-Docent  in  Midwifery  and  Gynecology  in  the  University 
of  Berlin.  Translated  from  the  Fourth  German  Edition  and  Edited  by  JOHN  W. 
TAYLOR,  F.R.C.S.,  Surgeon  to  the  Birmingham  and  Midlands  Hospital  for  Women  ; 
Vice-President  of  the  British  Gynecological  Society;  and  FREDERICK  EDGE,  M.D., 
M.R.C.P.,  F.R.C.S.,  Surgeon  to  the  Wolverhampton  and  District  Hospital  for  Women. 
With  105  Illustrations.  I2mo.  Cloth,  $1.50 

Dulles.     What  to  Do  First  In  Accidents  and  Poisoning. 

By  C.  W.  DULLES,  M.D.,  Surgeon  to  the  Rush  Hospital ;  formerly  Assistant  Surgeon 
2d  Regiment  N.  G.  Pa.,  etc.  Fifth  Edition,  Enlarged.  With  new  Illustrations. 
I2mo.  Cloth,  $1.00 

Edgar.     The  Practice  of  Obstetrics. 

By  J.  CLIFTON  EDGAR,  M.D.,  Professor  of  Obstetrics  Medical  Department  of  Cornell 
University,  New  York  City  ;  Physician  to  Mothers'  and  Babies'  Hospital,  and  to  the 
Emergency  Hospital,  etc.  With  many  Illustrations,  a  large  number  of  which  are 
Original.  Octavo.  In  Press. 

Emery.     A  Handbook  of  Bacteriological  Diagnosis. 

By  W.  D'ESTE  EMERY,  M.D.,  B.SC.  Lond.,  Lecturer  in  Pathology  and  Bacteriology  in 
the  University  of  Birmingham.  With  two  colored  plates  and  32  other  illustrations. 
Just  Ready.  '  Cloth,  $1.50 

Fagge.     Practice  of  Medicine. 

A  Text-Book  of  Medicine  by  the  late  C.  HILTON  FAGGE,  M.D.  Fourth  Edition, 
Revised  and  Edited  by  P.  H.  PYE-SMITH,  M.D.,  F.R.S.,  F.R.C.P.,  Consulting  Physi- 
cian to  Guy's  Hospital,  London,  etc.  Two  Vols.  Svo.  Vol.  I,  Just  Ready. 

Cloth,  $6.00 
Vol.  II,  Nearly  Ready. 

Fick.     Diseases  of  the  Eye  and  Ophthalmoscopy. 

A  Handbook  for  Physicians  and  Students.  By  DR.  EUGEN  FICK,  University  of 
Zurich.  Authorized  Translation  by  A.  B.  HALE,  M.D.,  Ophthalmic  Surgeon  United 
Hebrew  Charities  ;  Consulting  Ophthalmic  Surgeon  Charity  Hospital,  Chicago  ;  late 
Vol.  Assistant  Imperial  Eye  Clinic,  University  of  Kiel.  With  a  Glossary  and  158 
Illustrations,  many  of  which  are  in  Colors.  Svo. 

Cloth,  $4.50;  Sheep,  $5.50;  Half  Russia,  $6.50 

Fillebrown.     A  Text-Book  of  Operative  Dentistry. 
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FILLEBROWN,  M.D.,  D.M.D.,  Professor  of  Operative  Dentistry  in  the  Dental  School  of 
Harvard  University  ;  Member  of  the  American  Dental  Association,  etc.     Illustrated. 
Svo.  Cloth,  $2.25 

Fowler's  Dictionary  of  Practical  Medicine. 

By  Various  Writers.  An  Encyclopedia  of  Medicine.  Edited  by  JAMES  KINGSTON 
FOWLER,  M.A.,  M.D.,  F.R.C.P.,  Senior  Assistant  Physician  to,  and  Lecturer  on  Patho- 
logical Anatomy  at,  the  Middlesex  Hospital,  London.  Svo. 

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Surgical  Nursing. 

Comprising  the  Regular  Course  of  Lectures  upon  Abdominal  Surgery,  Gyne- 
cology,  and  General  Surgical  Conditions  delivered  at  the  Training  School  of 
the  Woman's  Hospital,  Philadelphia.  Third  Edition,  Revised.  69  Illustrations. 
1 2 mo.  Cloth,  $1.00 

Gardner.     The  Brewer,  Distiller,  and  Wine  Manufacturer. 

A  Handbook  for  all  interested  in  the  Manufacture  and  Trade  of  Alcohol  and  its 
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Goodall  and  Washbourn.     A  Manual  of  Infectious  Diseases. 

By  EDWARD  W.  GOODALL,  M.D.  (London),  Medical  Superintendent  Eastern  (Fever) 
Hospital,  Homerton,  London,  etc.  ;  and  J.  W.  WASHBOURN,  F.R.C.P.,  Assistant 
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ted with  Charts,  Diagrams,  and  Full-page  Plates.  Cloth,  $3.00 

Gould.      The    Illustrated    Dictionary    of    Medicine,    Biology,    and 
Allied  Sciences.     Fifth  Edition. 

Being  an  Exhaustive  Lexicon  of  Medicine  and  those  Sciences  Collateral  to  it : 
Biology  (Zoology  and  Botany),  Chemistry,  Dentistry,  Pharmacology,  Microscopy, 
etc.  By  GEORGE  M.  GOULD,  A.M.,  M.D.,  Editor  of  American  Medicine ;  President, 
1893-94,  American  Academy  of  Medicine,  etc.  With  many  Useful  Tables  and  numer- 
ous tFine  Illustrations.  Large  Square  Octavo.  1633  pages.  Fifth  Edition. 

Full  Sheep  or  Half  Dark-Green  Leather,  $10.00 
With  Thumb  Index,  $n.oo;  Half  Russia,  Thumb  Index,  $12.00 

"Few  persons  read  dictionaries  as  Theophile  Gautier  did — for  pleasure;  if,  however,  all 
dictionaries  were  as  readable  as  the  one  under  consideration,  his  taste  for  this  kind  of  literature 
would  be  less  s'ingular.  .  .  The  book  is  excellently  printed,  and  the  illustrations  are  admir- 

ably executed.  The  binding  is  substantial  and  even  handsome,  but  the  business-like  '  get-up  '  of 
the  book  makes  it  well  fitted  for  use  as  well  as  for  the  adornment  of  a  book-shelf." — The  British 
Medical  Journal,  London. 

The  Student's  Medical  Dictionary.     Eleventh  Ed.     Illustrated. 

Enlarged.  Including  all  the  Words  and  Phrases  generally  used  in  Medicine, 
with  their  proper  Pronunciations  and  Definitions,  based  on  Recent  Medical 
Literature.  With  Tables  of  the  Bacilli,  Micrococci,  Leukomains,  Ptomains; 
etc.,  of  the  Arteries,  Muscles,  Nerves,  Ganglia,  and  Plexuses  ;  Mineral  Springs 
of  the  U.  S.,  etc.,  and  a  new  Table  of  Eponymic  Terms  and  Tests.  Rewritten, 
Enlarged,  and  Improved.  With  many  Illustrations.  Small  octavo.  840  pages. 

Half  Morocco,  $2.50;  Thumb  Index,  $3.00 

"  One  pleasing  feature  of  the  book  is  that  the  reader  can  almost  invariably  find  the  definition 
under  the  word  he  looks  for,  without  being  referred  from  one  place  to  another,  as  is  too  commonly 
the  case  in  medical  dictionaries.  The  tables  of  the  bacilli,  micrococci,  leukomains,  and  ptomains 
are  excellent,  and  contain  a  large  amount  of  information  in  a  limited  space.  The  anatomical  tables 
are  also  concise  and  clear.  .  .  .  We  should  unhesitatingly  recommend  this  dictionary  to  our 
readers,  feeling  sure  that  it  will  prove  of  much  value  to  them." — The  American  Journal  of 
Medical  Science. 


16  P.  BLAKISTON'S  SON  &*   CO.' S 

Gould.    The  Pocket  Pronouncing  Medical  Lexicon.    Fourth  Edition. 

(30,000  Medical  Words  Pronounced  and  Defined.) 
A  Student's  Pronouncing  Medical  Lexicon.  Containing  all  the  Words,  their  Defini- 
tions and  Pronunciations,  that  the  Student  generally  comes  in  contact  with  ;  also 
elaborate  Tables  of  the  Arteries,  Muscles,  Nerves,  Bacilli,  etc.,  etc.;  a  Dose  List  in 
both  English  and  Metric  Systems,  a  new  table  of  Clinical  Eponymic  Terms,  etc., 
arranged  in  a  most  convenient  form  for  reference  and  memorizing.  Thin  641110. 
(6x3^  inches.)  838  pages.  The  System  of  Pronunciation  used  in  this  book  is  very 
simple.  A  New  Edition. 

Full  Limp  Leather,  Gilt  Edges,  $1.00  ;  With  Thumb  Index,  $1.25 

"  This  '  Dictionary  '  is  admirably  suited  to  the  uses  of  the  lecture-room,  or  for  the  purposes  of 

a  medical  defining  vocabulary — many  of  the  words  not  yet  being  found  in  any  other  dictionary, 

large  or  small,  while  all  of  the  words  are  those  of  the  living  medical  .literature  of  the  day." — The 

Virginia  Medical  Monthly. 

*#*  140,000  copies  of  Gould's  Dictionaries  have  been  sold. 
Sample  pages  and  descriptive  circulars  of  Gould' s  Dictionaries  free  upon  application. 

Borderland  Studies. 

Miscellaneous  Addresses  and  Essays  Pertaining  to  Medicine  and  the  Medical 
Profession,  and  their  Relations  to  General  Science.   350  pages.   I2mo.  Cloth,  $2.00 

Gould  and  Pyle.     Cyclopedia   of  Practical    Medicine  and   Surgery. 

72  Special  Contributors.     Illustrated.     One  Volume. 
A  Concise  Reference  Handbook,   Alphabetically  Arranged,    of  Medicine,  Surgery, 
Obstetrics,  Materia  Medica,  Therapeutics,  and  the  various  specialties,  with  Particular 
Reference  to  Diagnosis  and  Treatment.     Compiled  under  the  Editorial  Supervision 
of  DRS.  GEORGE  M.  GOULD  and  W.  L.  PYLE.     With  many  Illustrations. 
Large  Square  Octavo.     Uniform  with  Gould' s  ' '  Illustrated  Dictionary. ' ' 

Full   Sheep  or  Half  Dark-Green  Leather,  $10.00  ;  With  Thumb  Index,  $11.00 

Half  Russia,  Thumb  Index,  $12.00 

*#*  The  great  success  of  Dr.  Gould' s  ' '  Illustrated  Dictionary  of  Medicine ' '  sug- 
gested the  preparation  of  this  companion  volume,  which  should  be  to  the  physician  the 
same  trustworthy  handbook  in  the  broad  field  of  general  information  that  the  Dictionary 
is  in  the  more  special  one  of  the  explanation  of  words  and  the  statement  of  facts.  The 
aimjhas  been  to  provide  in  a  one-volume  book  all  the  material  usually  contained  in  the 
large  systems  and  much  which  they  do  not  contain.  Instead  of  long,  discursive  papers 
on  special  subjects  there  are  short,  concise,  pithy  articles  alphabetically  arranged,  giv- 
ing the  latest  methods  of  diagnosis,  treatment,  and  operating — a  working  book  in  which 
the  editors  and  their  collaborators  have  condensed  all  that  is  essential  from  a  vast 
amount  of  literature  and  personal  experience. 

The  seventy-two  special  contributors  have  been  selected  from  all  parts  of  the 
country  in  accordance  with  their  fitness  for  treating  special  subjects  about  which  they 
may  be  considered  expert  authorities.  They  are  all  men  of  prominence,  teachers, 
investigators,  and  writers  of  experience,  who  give  to  the  book  a  character  unequaled  by 
any  other  work  of  the  kind. 

At  each  reprinting  this  Cyclopedia  is  carefully  revised  and  augmented  so  as  to  in- 
clude important  innovations  and  in  order  to  keep  it  up-to-date. 

"The  book  is  a  companion  volume  to  Gould's  'Illustrated  Dictionary  of  Medicine,'  which 
every  physician  should  possess.  With  these  two  books  in  his  library,  every  busy  physician  will  save 
a  vast  amount  of  time  in  having  at  hand  an  instant  reference  cyclopedia  covering  every  subject  in 
surgery  and  medicine." — Chicago  Medical  Recorder. 

Pocket  Cyclopedia  of  Medicine  and  Surgery. 

Based  upon  Gould  and  Pyle's  Cyclopedia  of  Practical  Medicine  and  Surgery. 
Uniform  with  Gould's  Pocket  Dictionary. 

Full  Limp  Leather,  Gilt  Edges,  $1.00;  With  Thumb  Index,  $1.25 

See  next  page  for  List  of  Contributors. 


MEDICAL   AXD   SCIENTIFIC  PUBLICATIONS. 


17 


Gould   and   Pyle's   Cyclopedia  of  Medicine 
LIST  OF  CONTRIBUTORS 


Samuel  W.  Abbott,  A.M.,  M.D.,  Boston. 

James  M.  Anders,  M.D.,  LL.D.,  Pbila. 

Joseph  D.  Bryant,  M.D.,  New  York. 

James  B.  Bullitt,  M.D.,  Louisville. 

Charles  H.  Burnett,  A.M.,  M.D.,  Phila. 

J.  Abbott  Cantrell,  M.D.,  Philadelphia. 

Archibald  Church,  M  D.,  Chicago. 

L.  Pierce  Clark,  M.D.,  Sonyea,  N.  Y. 

Solomon  Solis-Cohen,  M.D.,  Philadelphia. 

Nathan  S.  Davis,  Jr.,  M.D.,  Chicago. 

Theodore  Diller,  M.D.,  Pittsburg. 

Augustus  A.  Eshner,  M,D.,  Philadelphia. 

J.  T.  Eskridge,  M.D.,  Denver,  Col. 

J.  McFadden  Gaston,  A.B.,  M.D.,  Atlanta, 
Ga. 

J.  McFadden  Gaston,  Jr.,  A.M.,  M.D.,  At- 
lanta, Ga. 

Virgil  P.  Gibney,  M.D.,  New  York. 

George  M.  Gould,  A.M.,  M.D.,  Phila. 

W.  A.  Hardaway,  A.M.,  M.D.,  St.  Louis. 

John  C.  Hemmeter,  M.B.,  M.D.,  Baltimore. 

Barton  Cooke  Hirst,  M.D.,  Philadelphia. 

Bayard  Holmes,  M.D.,  Chicago. 

Orville  Horwitz,  B.S.,  M.D.,  Philadelphia. 

Daniel  E.  Hughes,  M.D.,  Philadelphia. 

James  Nevins  Hyde,  A.M.,  M.D.,  Chicago. 

E.  Fletcher  Ingals,  A.M.,  M.D.,  Chicago. 

Abraham  Jacobi,  M.D.,  New  York. 

William  W.  Johnston,  M.D.,  Washington, 
D.  C. 

Wyatt  Johnston,  M.D.,  Montreal. 

Allen  A.Jones,  M.D.,  Buffalo. 
William  W.  Keen,  M.D.,  LL.D.,  Phila. 
Howard  S.  Kinne,  M.D.,  Philadelphia. 
Ernest  Laplace,  M.D.,  Philadelphia. 
Benjamin  Lee,  M.D.,  Philadelphia. 
Charles  L.  Leonard,  M.D.,  Philadelphia. 
James  Hendrie  Lloyd,  A.M.,  M.D.,  Phila. 
J.  W.  MacDonald,  M.D.  (Edin.),  F.R.C.S. 

Ed.,  Minneapolis. 
L.  S.  McMurtry,  M.D.,  Louisville. 
G.  Hudson  Makuen,  Philadelphia. 


Matthew  D.  Mann,  M.D.,  Buffalo. 

Henry    O.    Marcy,   A.M.,    M.D.,    LL.D., 

Boston. 

Rudolph  Matas,  M.D.,  New  Orleans. 
Joseph  M.  Mathews,  M.D.,  Louisville. 
John  K.  Mitchell,  M.D.,  Philadelphia. 
Harold  N.  Moyer,  M.D.,  Chicago. 
John  H.  Musser,  M.D.,  Philadelphia. 
A.  G.  Nicholls,  M.D.,  Montreal. 

A.  H.     Ohmann-Dusmesnil,     M.D.,     St. 
Louis. 

William  Osier,  M.D.,  Baltimore. 

Samuel   O.    L.  Potter,  A.M.,  M.D.,  M.R. 

C.P.  (London),  San  Francisco. 
Walter  L.  Pyle,  A.M.,  M.D.,  Philadelphia. 

B.  Alexander  Randall,  A.M.,  M.D.,  Phila. 
Joseph  Ransohoff,  M.D.,  F.R.C.S.  (Eng.), 

Cincinnati. 

Jay  F.  Schamberg,  A.M.,  M.D.,  Phila. 
Nicholas  Senn,  M.D.,  LL.D.,  Chicago. 
Richard  Slee,  M.D.,  Swiftwater,  Pa. 
S.    E.    Solly,   M.D.,    M.R.C.S.,    Colorado 

Springs,  Col. 

Edmond  Souchon,  M.D.,  New  Orleans. 
Ward  F.  Sprenkel,  M.D.,  Philadelphia. 
Charles  G.  Stockton,  M.D.,  Buffalo. 
John  Madison  Taylor,  A.M.,  M.D.,  Phila. 
William  S.  Thayer,  M.D.,  Baltimore. 
James  Thorington,  A.M.,  M.D.,  Phila. 
Martin  B.  Tinker,  M.D.,  Philadelphia. 
James  Tyson,  M.D.,  Philadelphia. 
J.  Hilton  Waterman,  M.D.,  New  York. 
H.  A.  West,  M.D.,  Galveston,  Texas. 
J.  William  White,  M.D.,  PH.D.,  Phila. 
Reynold  W.  Wilcox,  M.A.,  M.D.,  LL.D., 

New  York. 

George  Wilkins,  M.D.,  Montreal. 
DeForest  Willard,  M.D.,  Philadelphia. 
Alfred  C.  Wood,  M.D.,  Philadelphia. 
Horatio  C.  Wood,  M.D.,  LL.D.,  Phila. 
Albert  Woldert,  PH.G.,  M.D.,  Phila. 
James  K.  Young,  M.D.,  Philadelphia. 


"  It  is  difficult  to  describe  the  volume  before  us,  and  one  must  imagine  all  that  is  clinical 
at  the  present  day  as  being  briefly  and  yet  sufficiently  set  forth  under  an  alphabetical 
arrangement,  with  frequent  illustrations,  with  many  formulae  and  diagnostic  distinctions,  and 
with  perfect  homogeneity  ;  then  he  will  have  a  fair  picture  of  the  work.  We  feel  sure,  however, 
that  many  of  our  readers  will  make  the  better  acquaintance  of  the  book  by  becoming  its  possessors, 
and  we  commend  it  to  them  without  hesitation.  We  have  yet  to  find  wherein  it  is  erroneous  or 
disappointing,  and  we  regard  it  as  of  unlimited  value  to  the  average  medical  man." — The 
New  York  Medical  Journal. 

*#*  Sample  pages  and  description  upon  application. 


18  P.  BLAKISTON'S  SON  6-   CO.' S 

Gould  and  Pyle.     Compend  of  Diseases  of  the  Eye. 

Including  Refraction  Treatment  and  Operations,  with  a  Section  on  Local  Therapeutics. 
With  Formulae,  Glossary,  and  several  Tables.  By  DRS.  GEORGE  M.  GOULD  and 
W.  L.  PYLE.  Second  Edition.  109  Illustrations,  several  of  which  are  Colored. 
No.  8  ? Quiz- Compend?  Series.  Cloth,  80.;  Interleaved  for  Notes,  $1.00 

Gordinier.     The  Gross  and  Minute  Anatomy  of  the  Central  Nervous 

System.     261  Illustrations. 

By  H.  C.  GORDINIER,  A.M.,  M.D.,  Professor  of  Physiology  and  of  the  Anatomy  of 
the  Nervous  System  in  the  Albany  Medical  College  ;  Member  American  Neurological 
Association.  With  48  Full-page  Plates  and  213  other  Illustrations,  a  number  of 
which  are  printed  in  Colors  and  many  of  which  are  original.  Large  8vo. 

Handsome  Cloth,  $6.00  ;  Sheep,  $7.00  ;  Half  Russia,  $8.00 

"  This  is  an  excellent  book  on  a  fascinating  subject,  and  the  author  deserves  the  thanks  of  the 
English-speaking  medical  world  for  his  labor  in  getting  it  up.  There  are  works  enough  on  general 
anatomy,  and  dry  enough  they  are,  as  we  all  remember  only  too  well ;  but  the  anatomy  of  the 
nervous  system  alone  is  another  matter  entirely,  for' it  is  one  of  the  most  interesting  of  all  subjects 
•of  medical  study  at  the  same  time  that  it  is  one  of  the  most  difficult. "-r-The  Medical  Record,  N.  Y. 

Gorgas'  Dental  Medicine. 

A  Manual  of  Dental  Materia  Medica  and  Therapeutics.  By  FERDINAND  J.  S.  GORGAS, 
M.D.,  D.D.S.,  Professor  of  the  Principles  of  Dental  Science,  Oral  Surgery,  and  Dental 
Mechanism  in  the  Dental  Department  of  the  University  of  Maryland.  Seventh 
Edition,  Revised  and  Enlarged,  with  many  Formulae.  8vo.  Just  Ready. 

Cloth,  $4.00  ;  Sheep,  $5.00  ;  Half  Russia,  $6.00 

Questions  and  Answers. 

Embracing  the  Curriculum  of  the  Dental  Student.  Divided  into  three  parts. 
By  FERDINAND  J.  S.  GORGAS,  A.M.,  M.D.,  D.D.S.,  Author  of  "  Dental  Medicine," 
Editor  of  "Harris'  Principles  and  Practice  of  Dentistry  "  and  "Harris'  Dictionary 
of  Medical  Terminology  and  Dental  Surgery,"  Professor  of  the  Principles  of 
Dental  Science,  Oral  Surgery,  etc.,  in  the  University  of  Maryland,  Dental 
Department,  Baltimore,  Octavo.  Just  Ready.  Cloth,  $6.00 

Gray.     A  Treatise  of  Physics. 

By  ANDREW  GRAY,  LL.D.,  F.R.S.,  Professor  of  Natural  Philosophy  in  the  University 
of  Glasgow.  In  Three  Volumes. 

Vol.  I.     DYNAMICS   AND   PROPERTIES   OF  MATTER.     350  Illustrations.     Octavo. 
688  pages.  Cloth,  $4. 50 

Greeff.     The  Microscopic  Examination  of  the  Eye. 

By  Professor  R.  GREEFF.  Surgeon  to  the  Ophthalmic  Department  of  the  Royal  Charite" 
Hospital,  Berlin.  Translated  from  the  Second  German  Edition  by  HUGH  WALKER, 
M.A.,  M.D.,  Assistant  Surgeon  and  Pathologist  to  the  Ophthalmic  Department  of  the 
Glasgow  Royal  Infirmary.  I2mo.  Just  Ready.  Cloth,  $1.25 

Greene.     The  Medical  Examination  for  Life  Insurance 

and  its  Associated  Clinical  Methods.  With  Chapters  on  the  Insurance  of  Sub- 
standard Risks  and  Accident  Insurance.  By  CHARLES  LYMAN  GREENE,  M.D.,  of  St. 
Paul,  Clinical  Professor  of  Medicine  and  Physical  Diagnosis  in  the  University  of  Min- 
nesota. With  99  Illustrations,  many  of  which  are  original,  several  being  printed 
in  Colors.  Octavo.  Cloth,  $4.00 

Griffith's  Graphic  Clinical  Chart. 

Designed  by  J.  P.  CROZER  GRIFFITH,  M.D.,   Instructor  in  Clinical  Medicine  in  the 
University  of  Pennsylvania.    Sample  copies  free.    Put  up  in  loose  packages  of  50,  .  50 
Price  to  Hospitals:   500  copies,  $4.00  ;   1000  copies,  $7.50. 


MEDICAL  AND   SCIENTIFIC  PUBLICATIONS.  19 

Groff.      Materia  Medica  for  Nurses. 

With  Questions  for  Self-examination.  By  JOHN  E.  GROFF,  Pharmacist  to  the  Rhode 
Island  Hospital,  Providence.  Second  Edition,  Revised  and  Improved.  I2mo. 
Just  Ready.  Cloth,  $1.25 

Groves  and  Thorp.     Chemical  Technology. 

A  New  and  Complete  Work.     The  Application  of  Chemistry  to  the  Arts  and  Manu- 
factures.    Edited  by  CHARLES  E.  GROVES,  F.R.S.,  and  WM.  THORP,  B.SC.,  F.I.C., 
assisted  by  many  experts.    With  numerous  Illustrations.     Each  volume  sold  separately . 
Vol.      I.     FUEL  AND  ITS  APPLICATIONS.     607  Illustrations  and  4  Plates.     Octavo. 

Cloth,  $5.00;   yz  Mor.,  $6.50 

Vol.  II.  LIGHTING.  Candles,  Oils,  Lamps,  etc.  By  W.  Y.  DENT,  L.  FIELD, 
BOVERTON  REDWOOD,  and  D.  A.  Louis.  Illustrated. 
Octavo.  Cloth,  $4.00;  yz  Mor.,  15.50 

Vol.  III.  GAS  LIGHTING.  By  CHARLES  HUNT,  Manager  of  the  Birmingham 
Gasworks.  Illustrated.  Octavo. 

Cloth,  $3.50;   y2  Mor.,  $4.50 

Vol.  IV.  ELECTRIC  LIGHTING  AND  PHOTOMETRY.  By  ARTHUR  G.  COOKE,  M.A. 
(Cantab.),  Lecturer  on  Physics  and  Electric  Engineering 
at  the  Battersea  (London)  Polytechnic  ;  and  W.  J.  DIBDIN, 
F.I.C.,  F.C.S.,  late  Chemist  and  Superintending  Gas  Ex- 
aminer, London  County  Council.  In  Press. 

Gowers.     Manual  of  Diseases  of  the  Nervous  System. 
A  Complete  Text-Book.     By  SIR  WILLIAM  R.  GOWERS,  M.D.,  F.R.S.,  Physician  to 
National  Hospital  for  the  Paralyzed  and  Epileptic  ;  Consulting  Physician,  University 
College  Hospital ;  formerly  Professor  of  Clinical  Medicine,   University  College,  etc. 
Revised  and  Enlarged.     With  many  new  Illustrations.     Two  volumes.     Octavo. 

Vol.    I.     Diseases  of  the  Nerves  and  Spinal  Cord. 

Third  Edition.  Cloth,  $4.00  ;  Sheep,  $5.00  ;  Half  Russia,  $6.00 

Vol.  II.      Brain  and   Cranial   Nerves ;    General   and    Functional 

Diseases. 

Second  Edition.  Cloth,  $4.00;  Sheep,  15.00;  Half  Russia,  $6.00 

*#*  This  book  has  been  translated  into  German,  Italian,  and  Spanish.     It  is  pub- 
lished in  London,  Milan,  Bonn,  Barcelona,  and  Philadelphia. 

Syphilis  and  the  Nervous  System. 

Being  a  Revised  Reprint  of  the  Lettsomian  Lectures  for  1890,  delivered  before 
the  Medical  Society  of  London.      I2mo.  Cloth,  $1.00 

Epilepsy  and  Other  Chronic  Convulsive  Diseases. 

Their  Causes,  Symptoms,  and  Treatment     Second  Edition.  Cloth,  $3.00 

Hadley.     General  Medical  and  Surgical  Nursing. 

A  Manual  for  Nurses.     By  DR.  W.  G.   HADLEY,    Physician  to,  and  Lecturer  on 

Medicine  to  the  Nurses  at,  the  London  Hospital.     With  an  Appendix  on  Sick-Room 

Cookery.      I2mo.      326  pages.  Cloth,  $1.25 

Haig.     Causation  of  Disease  by  Uric  Acid.     Fifth  Edition. 

A  Contribution  to  the  Pathology  of  High  Arterial  Tension,  Headache,  Epilepsy, 
Mental  Depression,  Gout,  Rheumatism,  Diabetes,  Bright' s  Disease,  Anaemia,  etc. 
By  ALEXANDER  HAIG,  M.A.,  M.D.  (Oxon.),  F.R.C.P.,  Physician  to  Metropolitan  Hos- 
pital, London.  75  Illustrations.  Fifth  Edition.  8vo.  846  pages.  Cloth,  £3.00 

Diet  and  Food. 

Considered  in  Relation  to  Strength  and  Power  of  Endurance.     Third  Edition, 
Revised.  Cloth,  |i.oo 


20  P.  BLAKISTON'S  SON  &   CO.' S 


Hall.     Diseases  of  the  Nose  and  Throat. 

By  F.  DE  HAVILLAND  HALL,  M.D.,  F.R.C.P.  (Lond.),  Physician  to  the  Westminster 
Hospital ;  President  of  the  Laryngological  Society  of  London  ;  Joint  Lecturer  on  the 
Principles  and  Practice  of  Medicine  at  the  Westminster  Hospital ;  and  HERBERT 
TILLEY,  M.D.,  B.S.  (Lond.),  F.R.C.S.  (Eng.),  Surgeon  to  the  Throat  Hospital,  Golden 
Square  ;  Lecturer  on  Diseases  of  the  Nose  and  Throat,  London  Post-Graduate  College 
and  Poly  clinic.  Second  .Edition,  Thoroughly  Revised,  with  2  Plates  and  80  Illustra- 
tions. Just  Ready.  Cloth,  $2.75 

Hamilton.     Lectures  on  Tumors 

from  a  Clinical  Standpoint.  By  JOHN  B.  HAMILTON,  M.D.,  LL.D.,  late  Professor  of 
Surgery  in  Rush  Medical  College,  Chicago  ;  Professor  of  Surgery,  Chicago  Polyclinic  ; 
Surgeon  Presbyterian  Hospital,  etc.  Third  Edition,  Revised.  With  New  Illustra- 
tions. I2mo.  Cloth,  $1.25 

Hansell  and  Reber.     Muscular  Anomalies  or  the  Eye. 

By  HOWARD  F.  HANSELL,  A.M.,  M.D.,  Clinical  Professor  of  Ophthalmology,  Jefferson 
Medical  College  ;  Professor  of  Diseases  of  the  Eye,  Philadelphia  Polyclinic,  etc. ;  and 
WENDELL  REBER,  M.D.,  Instructor  in  Ophthalmology,  Philadelphia  Polyclinic,  etc. 
With  i  Plate  and  28  other  Illustrations.  I2mo.  Cloth,  $1.50 

Hansell  and  Bell.     Clinical  Ophthalmology. 

By  HOWARD  F.  HANSELL,  A.M.,  M.D.,  and  JAMES  H.  BELL,  M.D.  With  Colored  Plate 
of  Normal  Fundus  and  120  Illustrations.  I2mo.  Cloth,  $1.50 

Hare.     Mediastinal  Disease. 

The  Pathology,  Clinical  History,  and  Diagnosis  of  Affections  of  the  Mediastinum 
other  than  those  of  the  Heart  and  Aorta.  By  H.  A.  HARE,  M.D.,  Professor  of 
Materia  Medica  and  Therapeutics  in  Jefferson  Medical  College.  8vo.  Illustrated. 

Cloth,  $2.00 

Harlan.     Eyesight 

and  How  to  Care  for  It.  By  GEORGE  C.  HARLAN,  M.D.,  Professor  of  Diseases  ol 
the  Eye,  Philadelphia  Polyclinic.  Illustrated.  '  Cloth,  .40 

Harris'  Principles  and  Practice  of  Dentistry. 

Including  Anatomy,  Physiology,  Pathology,  Therapeutics,  Dental  Surgery,  and 
Mechanism.  By  CHAPIN  A.  HARRIS,  M.D.,  D.D.S.,  late  President  of  the  Baltimore 
Dental  College;  Author  of  "Dictionary  of  Medical  Terminology  and  Dental  Sur- 
gery." Thirteenth  Edition,  Revised  and  Edited  by  FERDINAND  J.  S.  GORGAS, 
A.M.,  M.D.,  D.D.S.,  Author  of  "Dental  Medicine;"  Professor  of  the  Principles  of 
Dental  Science,  Oral  Surgery,  and  Dental  Mechanism  in  the  University  of  Maryland. 
1250  Illustrations.  II 80  pages.  8vo. 

Cloth,  $6.00;  Leather,  $7.00;  Half  Russia,  $8.00 

Dictionary  of  Dentistry. 

Including  Definitions  of  such  Words  and  Phrases  of  the  Collateral  Sciences  as 
Pertain  to  the  Art  and  Practice  of  Dentistry.  Sixth  Edition,  Rewritten,  Re- 
vised, and  Enlarged.  By  FERDINAND  J.  S.  GORGAS,  M.D.,  D.D.S.,  Author  of 
"  Dental  Medicine  ;"  Editor  of  Harris'  "  Principles  and  Practice  of  Dentistry  ;" 
Professor  of  Principles  of  Dental  Science,  Oral  Surgery,  and  Prosthetic  Dentistry 

.;     in  the  University  of  Maryland.     Octavo.  Cloth,  $5.00  ;  Leather,  $6.00 


MEDICAL   AND   SCIENTIFIC  PUBLICATIONS.  21 

Hartridge.      Refraction. 

The  Refraction  of  the  Eye.  A  Manual  for  Students.  By  GUSTAVUS  HARTRIDGE, 
F.R.C.S.,  Senior  Surgeon  Royal  Westminster  Ophthalmic  Hospital;  Ophthalmic 
Surgeon  to  St.  Bartholomew's  Hospital,  etc.  105  Illustrations  and  Sheet  of  Test 
Types.  Eleventh  Edition,  Revised  and  Enlarged.  Just  Ready.  Cloth,  11.50 

On  the  Ophthalmoscope. 

A  Manual  for  Physicians  and  Students.     Fourth  Edition,  Revised.    With  Colored 
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Hartshorne.     Our  Homes. 

Their  Situation,  Construction,  Drainage,  etc.  By  HENRY  HARTSHORNE,  M.D.  Illus- 
trated. Cloth,  .40' 

Hatfield.     Diseases  of  Children. 

By  MARCUS  P.  HATFIELD,  Professor  of  Diseases  of  Children,  Chicago  Medical  Col- 
lege. With  a  Colored  Plate.  Second  Edition.  Being  No.  14  ?  Quiz- Comp  end  ? 
Series.  I2mo.  Cloth,  .80  ;  Interleaved  for  the  Addition  of  Notes,  #1.00 

"  Dr.  Hatfield  seems  to  have  most  thoroughly  appreciated  the  needs  of  students,  and  most 
excellently  has  he  condensed  his  matter  into  available  form.  It  is  in  accord  with  the  most  recent 
teachings,  and  while  brief  and  concise,  is  surprisingly  complete.  .  .  .  It  is  free  from  irritating 
repetition  of  questions  and  answers  which  mars  so  many  of  the  compends  now  in  use.  Written  in 
systematic  form,  the  consideration  of  each  disease  begins  with  its  definition,  and  proceeds  through 
the  usual  subheadings  to  prognosis  and  treatment,  thus  furnishing  a  complete,  readable  text-book." 
— Annals  of  Gynecoltgy  and  Pediatry. 

Heath.     Minor  Surgery  and  Bandaging. 

By  CHRISTOPHER  HEATH,  F.R.C.S.,  Holme  Professor  of  Clinical  Surgery  in  Univer- 
sity College,  London.  Twelfth  Edition,  Revised  and  Enlarged  by  BILTON  POLLARD, 
F.R.C.S.,  Surgeon  University  College  Hospital,  London.  With  195  Illustrations, 
Formulae,  Diet  List,  etc.  I2mo.  Cloth,  $1.50 

Practical  Anatomy. 
A  Manual  of  Dissections.     Eighth  London  Edition.     300  Illus.         Cloth,  $4.25 

Clinical  Lectures  on  Surgical  Subjects. 

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Hedley.     Therapeutic  Electricity  and  Practical  Muscle  Testing. 

By  W.  S.  HEDLEY,  M.D.,  M.R.C.S.,  in  charge  of  the  Electrotherapeutic  Department 
of  the  London  Hospital.  99  Illustrations.  Octavo.  Cloth,  $2.50 

Heller.     Essentials  of  Materia  Medica,  Pharmacy,  and  Prescription 

Writing. 

By  EDWIN  A.  HELLER,  M.D.,  Quiz-Master  in  Materia  Medica  and  Pharmacy  at  the 
Medical  Institute,  University  of  Pennsylvania.  I2mo.  Cloth,  $  1.50 

Henry.     Anaemia. 

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Philadelphia.  Half  Cloth,  .50 

Heusler.     The  Terpenes. 

By  FR.  HEUSLER,  PH.D.,  Privatdocent  of  Chemistry  in  the  University  at  Bonn. 
Authorized  Translation  and  Revision  by  F.  J.  Pond,  PH.D.,  Assistant  Professor  of 
Chemistry,  Pennsylvania  State  College.  Cloth,  $4.00 


22  P.   BLAKISTON'S  SON  &*    CO.' S 

Hemmeter.     Diseases  of  the  Stomach.     Third  Edition. 

Their  Special  Pathology,  Diagnosis,  and  Treatment.  With  Sections  on  Anatomy, 
Analysis  of  Stomach  Contents,  Dietetics,  Surgery  of  the  Stomach,  etc.  By  JOHN  C. 
HEMMETER,  M.D.,  PHILOS.D.,  Professor  in  the  Medical  Department  of  the  University 
of  Maryland  ;  Consultant  to  the  University  Hospital  ;  Director  of  the  Clinical  Labor- 
atory, etc. ;  formerly  Clinical  Professor  of  Medicine  at  the  Baltimore  Medical  College, 
etc.  Third  Edition,  Revised.  With  15  Plates  and  41  other  Illustrations,  some  of 
which  are  in  Colors.  Cloth,  $6.00;  Leather,  $7.00;  Half  Russia,  $8.00 

Diseases  of  the  Intestines. 

A  Complete  Systematic  Treatise  on  Diseases  of  the  Intestines,  including  their 
Special  Pathology,  Diagnosis,  and  Treatment,  with  Sections  on  Anatomy  and 
Physiology,  Microscopic  and  Chemic  Examination  of  the  Intestinal  Contents, 
Secretions,  Feces,  and  Urine.  Intestinal  Bacteria  and  Parasites  ;  Surgery  of  the 
Intestines  ;  Dietetics,  Diseases  of  the  Rectum,  etc.  With  many  Full-page  Plates, 
Colored  and  other  Illustrations,  most  of  which  are  Original.  2  vols.  Octavo. 
Just  Ready.  Vol.  I.  Cloth,  $5.00;  Sheep,  $6.00 

Vol.  II.     Cloth,  $5.00;  Sheep,  #6.00 

THE  SECTION  ON  ANATOMY  has  been  prepared  by  DR.  J.  HOLMES  SMITH,  Associate 
Professor  and  Demonstrator  of  Anatomy,  and  Lecturer  on  Clinical  Surgery,  University 
of  Maryland,  Baltimore.  THE  SECTION  ON  BACTERIA  OF  THE  INTESTINES  has  been 
prepared  by  DR.  WM.  ROYAL  STOKES,  Associate  Professor  of  Pathology  and  Bacteriology, 
and  Visiting  Pathologist  to  the  University  Hospital,  University  of  Maryland,  Baltimore. 
THE  SECTION  ON  DISEASES  OF  THE  RECTUM  has  been  prepared  by  DR.  THOMAS  C. 
MARTIN,  Professor  of  Proctology,  Cleveland  College  of  Physicians  and  Surgeons.  THE 
SECTION  ON  EXAMINATION  OF  URINE  AND  FECES  has  been  prepared  by  DR.  HARRY 
ADLER,  Demonstrator  of  Clinical  Pathology,  Associate  Professor  of  Diseases  of  the 
Stomach  and  Intestines,  University  of  Maryland,  Baltimore.  THE  ILLUSTRATIONS  form 
a  most  useful  and  practical  series  of-  pictures, — nearly  all  have  been  reproduced  from 
pathological  preparations  and  original  drawings,  a  few  being  printed  in  several  colors. 

Hewlett.     Manual  of  Bacteriology.     75  Illustrations. 

By  R.  T.  HEWLETT,  M.D.,  M.R.C.P.,  Assistant  Bacteriologist  British  Institute  of  Pre- 
ventive Medicine,  etc.  Second  Edition,  Revised.  Just  Ready.  Cloth,  $4.00 

Hollopeter.     Hay  Fever  and  Its  Successful  Treatment. 

By  W.  C.  HOLLOPETER,  A.M.,  M.D.,  Clinical  Professor  of  Pediatrics  in  the  Medico- 

¥  Chirurgical  College  of  Philadelphia  ;  Physician  to  the  Methodist  Episcopal,  Medico- 

Chirurgical,  and  St.  Joseph's  Hospitals,  etc.     Second  Edition.     I2mo.       Cloth,  $1.00 

Holden's  Anatomy.     Seventh  Edition. 

A  Manual  of  the  Dissections  of  the  Human  Body.  By  JOHN  LANGTON,  F.R.C.S., 
Surgeon  to,  and  Lecturer  on  Anatomy  at,  St.  Bartholomew's  Hospital.  Carefully 
Revised  by  A.  HEWSON,  M.D.,  Demonstrator  of  Anatomy,  Jefferson  Medical  College, 
Philadelphia,  etc.  320  Illustrations.  Two  small  compact  volumes.  I2mo. 

Vol.    I.     Scalp,  Face,  Orbit,  Neck,  Throat,  Thorax,  Upper  Extremity.     435  pages. 

153  Illustrations.  Oil  Cloth,  $1.50 

Vol.  II.     Abdomen,    Perineum,    Lower    Extremity,    Brain,    Eye,    Ear,    Mammary 

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Oil  Cloth,  $1.50 

Human  Osteology. 

Comprising  a  Description  of  the  Bones,  with  Colored  Delineations  of  the  Attach- 
ments of  the  Muscles.  The  General  and  Microscopical  Structure  of  Bone  and 
its  Development.  Eighth  Edition,  Carefully  Revised.  Edited  by  CHARLES 
STEWART,  F.R.S.,  and  R.  W.  REID,  M.D.,  F.R.C.S.  With  Colored  Lithographic 
Plates  and  Numerous  Illustrations.  Cloth,  $5.25 

Landmarks. 

Medical  and  Surgical.     Fourth  Edition.      8vo.  Cloth,  .75 


MEDICAL  AND   SCIENTIFIC  PUBLICATIONS.  23 

Holland.     The  Urine,  the  Gastric  Contents,  the  Common  Poisons, 

and  the  Milk.     Illustrated. 

Memoranda  (Chemical  and  Microscopical)  for  Laboratory  Use.  By  J.  W.  HOLLAND, 
M.D.,  Professor  of  Medical  Chemistry  and  Toxicology  in  Jefferson  Medical  College 
of  Philadelphia.  Sixth  Edition,  Enlarged.  Illustrated  and  Interleaved.  121110. 

,     r^  j      r  c  Cloth,  $1.00 

Horwitz  s  Compend  or  burgery. 

Including  Minor  Surgery,  Amputations,  Bandaging,  Fractures,  Dislocations,  Surgical 
Diseases,  etc.,  with  Differential  Diagnosis  and  Treatment.  By  ORVILLE  HORWITZ, 
B.S.,  M.D.,  Professor  of  Genito-Urinary  Diseases,  late  Demonstrator  of  Surgery, 
Jefferson  Medical  College.  Fifth  Edition.  167  Illustrations  and  98  Formulae.  I2mo. 
No.  9  ? Quiz- Compend?  Series.  Cloth,  .80  ;  Interleaved  for  Notes,  £1.00 

*#*  A  Spanish  translation  of  this  book  has  recently  been  published  in  Barcelona. 

Horsley.     The  Brain  and  Spinal  Cord, 

the  Structure  and  Functions  of.  By  VICTOR  A.  HORSLEY,  M.B.,  F.R.S.,  etc.,  As- 
sistant Surgeon  University  College  Hospital,  London,  etc.  Illustrated.  Cloth,  $2.50 

Hovell.     Diseases  of  the  Ear  and  Naso-Pharynx. 

A  Treatise  including  Anatomy  and  Physiology  of  the  Organ,  together  with  the  treat- 
ment of  the  affections  of  the  Nose  and  Pharynx  which  conduce  to  aural  disease.  By 
T.  MARK  HOVELL,  F.R.C.S.  (Edin.),  M.R.C.S.  (Eng.),  Aural  Surgeon  to  the  London 
Hospital  for  Diseases  of  the  Throat,  etc.  128  Illus.  Second  Edition.  Cloth,  $5.50 

Humphrey.     A  Manual  for  Nurses.     Twenty-third  Edition. 

Including  General  Anatomy  and  Physiology,  Management  of  the  Sick-room,  etc.  By 
LAURENCE  HUMPHREY,  M.A.,  M.B.,  M.R.C.S.,  Assistant  Physician  to  Addenbrook's 
Hospital,  Cambridge,  England.  231!  Edition.  I2mo.  79  Illustrations.  Cloth,  $1.00 

Hughes  and  Keith.     Dissections.     Illustrated. 

A  Manual  of  Dissections  by  ALFRED  W.  HUGHES,  M.B.,  M.R.C.S.  (Edin.),  late  Pro- 
fessor of  Anatomy  and  Dean  of  Medical  Faculty,  King's  College,  London,  etc.,  and 
ARTHUR  KEITH,  M.D.,  Lecturer  on  Anatomy,  London  Hospital  Medical  College,  etc. 
In  three  parts,  with  many  Colored  and  other  Illustrations. 

I.  Upper  and  Lower  Extremity.    38  Plates,  1 16  other  Illustrations.     Just  Ready. 

Cloth,  $3.00 

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tions.    Just  Ready.  Cloth,  $3.00 

Hughes.     Compend  of  the  Practice  of  Medicine.     Sixth  Edition. 

Giving  the  Synonyms,  Definition,  Causes,  Symptoms,  Pathology,  Prognosis,  Diag- 
nosis, Treatment,  etc.,  of  each  Disease.  The  Treatment  is  especially  full  and  a 
number  of  valuable  Prescriptions  have  been  incorporated.  Sixth  Edition,  Revised 
and  Enlarged.  By  DANIEL  E.  HUGHES,  M.D.,  Chief  Resident  Physician  Philadel- 
phia Hospital ;  formerly  Demonstrator  of  Clinical  Medicine  at  Jefferson  Medical 
College,  Philadelphia.  Being  Nos.  2  and 3  ? Quiz- Compend?  Series. 

Quiz-Compend  Edition,  in  two  Parts. 

PART  I. — Continued,  Eruptive,  and  Periodical  Fevers,  Diseases  of  the  Mouth, 
Stomach,  Intestines,  Peritoneum,  Biliary  Passages,  Liver,  Kidneys,  Blood,  etc., 
Parasites,  etc.,  and  General  Diseases,  etc. 

PART  II. — Physical  Diagnosis,  Diseases  of  the  Respiratory  System,  Circulatory 
Systejn,  Diseases  of  the  Brain  and  Nervous  System,  Mental  Diseases,  etc. 

Price  of  each  Part,  in  Cloth,  .80  ;  Interleaved  for  the  Addition  of  Notes,  $1.00 

Physicians'  Edition. 

In  one  volume,  including  the  above  two  parts,  a  Section  on  Skin  Diseases,  and 
an  Index.     Sixth  Revised  and  Enlarged  Edition.     625  pages. 

Full  Morocco,  Gilt  Edges,  Round  Corners,  $2.25 


24  P.   BLAKISTON'S  SON  6-    CO.' S 

Ireland.     The  Mental  Affections  of  Children. 

Idiocy,  Imbecility,  Insanity,  etc.  By  W.  W.  IRELAND,  M.D.  (Edin.),  of  the  Home 
and  School  for  Imbeciles,  Mavisbush,  Scotland  ;  Second  Edition,  Revised  and  En- 
larged. Cloth,  $4.00 

Jacoby.     Electrotherapy.      Illustrated. 
See  COHEN,  Physiologic  Therapeutics,  page  9. 

Jacobson.     The  Operations  of  Surgery. 

By  W.  H.  A.  JACOBSON,  F.R.C.S.  (Eng.),  Surgeon  Guy's  Hospital,  etc.,  and  F.  J. 
STEWARD,  F.R.C.S.,  Assistant  Surgeon  Guy's  Hospital  and  the  Hospital  for  Sick  Chil- 
dren, Great  Ormand  Street,  London.  With  550  Illustrations.  Fourth  Edition, 
Revised  and  Enlarged.  Two  volumes.  Octavo.  15 24  pages. 

Cloth,  $10.00;  Leather,  $12.00 

Jennings.     A  Manual  of  Ophthalmoscopy. 

By  J.  E.  JENNINGS,  M.D.  (Univ.  Penna.),  Formerly  Clinical  Assistant  Royal  London 
Ophthalmic  Hospital,  London  ;  Fellow  of  the  British  Laryngological  and  Rhinological 
Association  ;  Member  of  the  American  Medical  Association  ;  Member  of  the  St. 
Louis  Medical  Society,  etc.  With  95  Illustrations  and  I  Colored  Plate.  Just  Ready. 

Cloth,  $1.50 

Jones.     Medical  Electricity.     Third  Edition. 

A  Practical  Handbook  for  Students  and  Practitioners  of  Medicine.  By  H.  LEWIS 
JONES,  M.A.,  M.D.,  F.R.C.P.,  Medical  Officer  in  Charge  Electrical  Department,  St. 
Bartholomew's  Hospital.  Third  Edition  of  Steavenson  and  Jones'  Medical  Elec- 
tricity, Revised  and  Enlarged.  117  Illustrations.  532  pages.  I2mo.  Cloth,  $3.00 

Jones.     Outlines  of  Physiology. 

By  EDWARD  GROVES  JONES,  M.D.,  Assistant  Professor  of  Physiology  and  Pathological 
Anatomy,  Atlanta  College  of  Physicians  and  Surgeons.  96  Illustrations.  I2mo. 

Cloth,  $1.50 

Keay.     Gall-Stones. 

The  Medical  Treatment  of  Gall-Stones.  By  J.  H.  KEAY,  M.A.,  M.D.,  Physician  to 
Trinity  Hospital,  Greenwich,  London.  I2mo.  Just  Ready.  Cloth,  $1.25 

Keen.     Clinical  Charts. 

A  Series  of  Seven  Outline  Drawings  of  the  Human  Body,  on  which  may  be  marked 
the  course  of  any  Disease,  Fractures,  Operations,  etc.  By  W.  W.  KEEN,  M.D., 
Professor  of  the  Principles  of  Surgery  and  Clinical  Surgery,  Jefferson  Medical  College. 
Each  Drawing  may  be  had  separately  gummed  on  back  for  pasting  in  case  book. 
25  to  the  pad.  Price,  25  cents.  Special  Charts  wUl  be  printed  to  order.  Samples  free. 

Kehr.     Diagnosis  of  Gail-Stone  Disease. 

Including  one  hundred  Clinical  and  Operative  Cases  illustrating  Diagnostic  Points  of 
the  Different  Forms  of  the  Disease.  By  PROF.  DR.  HANS  KEHR,  of  Halberstadt. 
Authorized  Translation  by  WILLIAM  WOTKYNS  SEYMOUR,  A.B.  (Yale),  M.D.  (Harvard), 
of  Troy,  N.  Y.  I2mo.  370  pages.  Cloth,  $2.50 

Kenwood.     Public  Health  Laboratory  Work. 

By  H.  R.  KENWOOD,  M.B.,  D.P.H.,  F.C.S.,  Assistant  Professor  of  Public  Health, 
University  College,  London,  etc.  116  Illustrations  and  3  Plates.  Cloth,  $2.00 


MEDICAL   AND   SCIENTIFIC  PUBLICATIONS.  25 

Kirkes'  Physiology.     Seventeenth  Edition. 

(  The  only  Authorized  Edition.  I2mo.  Dark  Red  Cloth.'}  A  Handbook  of  Physiology. 
Sixteenth  London  Edition,  Revised  and  Enlarged.  By  W.  D.  HALLIBURTON,  M.D., 
F.R.S.,  Professor  of  Physiology,  King's  College,  London.  Thoroughly  Revised  and 
in  many  parts  Rewritten.  68 1  Illustrations,  a  number  of  which  are  printed  in  Colors. 
888  pages.  I2mo.  Cloth,  $3.00  ;  Leather,  $3.75 

IMPORTANT  NOTICE.     This  is  the  identical  Edition  of  "  Kirkes'  Physiology,"  as  published  in 

• London  by  John  Murray,  the  sole  owner  of  the  book,  and  containing 

the  revisions  and  additions  of  Dr.  Halliburton,  and  the  new  and  original  illustrations  included  at 
his  suggestion.     This  edition  has  been  carefully  and  thoroughly  revised. 

Kleen.     Diabetes  Mellitus  and  Glycosuria. 
Their  Diagnosis  and  Treatment.     By  DR.  EMIL  KLEEN.     Octavo.  Cloth,   $2.50 

Knight.     Diseases  of  the  Throat. 

A  Manual  for  Students.  By  CHARLES  H.  KNIGHT,  M.D.,  Professor  of  Laryngology, 
Cornell  University  Medical  College  ;  Surgeon  to  Throat  Department,  Manhattan  Eye 
and  Ear  Hospital,  etc.  Illustrated.  Nearly  Ready. 

Knopf.     Pulmonary  Tuberculosis.     Its  Modern  Prophylaxis  and  the 
Treatment  in  Special  Institutions  and  at  Home. 

By  S.  A.  KNOPF,  M.D.,  Physician  to  the  Lung  Department  of  the  New  York  Throat 
and  Nose  Hospital ;  former  Assistant  Physician  to  Professor  Dettweiler,  Falkenstein 
Sanatorium,  Germany,  etc.  Illustrated.  Octavo.  Cloth,  $3.00 

Landis'  Compend  of  Obstetrics. 

Especially  adapted  to  the  Use  of  Students  and  Physicians.  By  HENRY  G.  LANDIS, 
M.D.  Seventh  Edition,  Revised  by  WM.  H.  WELLS,  M.D.,  Demonstrator  of  Clinical 
Obstetrics,  Jefferson  Medical  College  ;  Member  Obstetrical  Society  of  Philadelphia, 
etc.  With  52  Illustrations.  No.  j  ?  Quiz- Comp  end?  Series.  Just  Ready. 

Cloth,  .80  ;  Interleaved  for  the  Addition  of  Notes,  $1.00 

Landois.     A  Text-Book  of  Human  Physiology. 

Including  Histology  and  Microscopical  Anatomy,  with  Special  Reference  to  the  Re- 
quirements of  Practical  Medicine.  By  DR.  L.  LANDOIS,  Professor  of  Physiology  and 
Director  of  the  Physiological  Institute  in  the  University  of  Greifswald.  Fifth  Ameri- 
can translated  from  the  last  German  Edition,  with  Additions,  by  WM.  STIRLING, 
M.D.,  D.SC.,  Brackenbury  Professor  of  Physiology  and  Histology  in  Owens  College, 
and  Professor  in  Victoria  University,  Manchester  ;  Examiner  in  Physiology  in  Uni- 
versity of  Oxford,  England.  With  845  Illustrations,  many  of  which  are  printed  in 
Colors.  8vo.  In  Press. 

Lazarus-Barlow.     General  Pathology. 

By  W.  S.  LAZARUS-BARLOW,  M.D.,  Demonstrator  of  Pathology  at  the  University  of 
Cambridge,  England.  795  pages.  Octavo.  Cloth,  $5.00 

Lee.     The  Microtomist's  Vade  Mecum.     Fifth  Edition. 
A  Handbook  of  the  Methods  of  Microscopic  Anatomy.     By  ARTHUR  BOLLES  LEE, 
formerly  Assistant  in  the  Russian  Laboratory  of  Zoology  at  Villefranche-sur-Mer  (Nice). 
894  Articles.     Enlarged,  Revised,  and  Rearranged.      532  pages.     8vo.     Cloth,  #4.00 

Leffmann  and  Beam.  Food  Analysis.  Illustrated. 
Select  Methods  in  Food  Analysis.  By  HENRY  LEFFMANN,  M.D.,  Professor  of  Chem- 
istry in  the  Woman's  Medical  College  of  Pennsylvania  and  in  the  Wagner  Free 
Institute  of  Science  ;  Pathological  Chemist,  Jefferson  Medical  College  Hospital,  Phila- 
delphia ;  Vice-President  (1901)  Society  Public  Analysts,  etc.;  and  WILLIAM  BEAM, 
A.M.  With  many  useful  Tables,  4  Plates  and  53  other  Illustrations.  I2mo. 

Cloth,  $2.50 


P.   BLAKISTON'S  SON  &    CO.' S 


Leffmann.     Compend  of  Medical  Chemistry. 

Inorganic  and  Organic.  Including  Urine  Analysis.  By  HENRY  LEFFMANN,  M.D., 
Professor  of  Chemistry  in  the  Woman's  Medical  College  of  Pennsylvania  and  in  the 
Wagner  Free  Institute  of  Science,  Philadelphia  ;  Pathological  Chemist  Jefferson  Medi- 
cal College  Hospital ;  Vice-President,  1901,  Society  of  Public  Analysts,  etc.  No.  10 
?  Quiz- Compend?  Series.  Fourth  Edition,  Rewritten. 

Cloth,  .80  ;  Interleaved  for  the  Addition  of  Notes,  $1.00 

The  Coal-Tar  Colors. 

With  Special  Reference  to  their  Injurious  Qualities  and  the  Restrictions  of  their 
Use.     A  Translation  of  Theodore  Weyl's  Monograph.      I2mo.         Cloth,  $1.25 

Examination  of  Water 

for  Sanitary  and  Technical  Purposes.     Fourth   Edition,  Enlarged.     Illustrated. 
I2mo.  Cloth,  $1.25 

Analysis  of  Milk  and  Milk  Products. 

Arranged  to  suit  the  needs  of  Analytical  Chemists,  Dairymen,  and  Milk  Inspec- 
tors.    Second  Edition,  Revised  and  Enlarged.     Illustrated.     i2mo.     Cloth,  $1.25 

Handbook  of  Structural  Formulae 

for  the  Use  of  Students,  containing  180  Structural  and  Stereo-chemic  Formulae. 
I2mo.     Interleaved.  Cloth,  $1.00 

Lewers.     On  the  Diseases  of  Women. 

A  Practical  Treatise.  By  DR.  A.  H.  N.  LEWERS,  Assistant  Obstetric  Physician  to 
the  London  Hospital.  146  Engravings.  Fifth  Edition,  Revised.  Cloth,  $2.50 

Lewis  (Bevan).      Mental  Diseases. 

A  Text-Book  having  Special  Reference  to  the  Pathological  Aspects  of  Insanity.  By 
BEVAN  LEWIS,  L.R.C.P.,  M.R.C.S.,  Medical  Director  West  Riding  Asylum,  Wake- 
field,  England.  26  Lithograph  Plates  and  other  Illustrations.  Second  Edition,  Re- 
vised and  Enlarged.  8vo.  Cloth,  $7.00 

Lincoln.     School  and  Industrial  Hygiene. 
By  D.  F.  LINCOLN,  M.D.  Cloth,  .40 

Longley's  Pocket  Medical  Dictionary. 

Giving  the  Definition  and  Pronunciation  of  Words  and  Terms  in  General  Use  in 
Medicine.  With  an  Appendix,  containing  Poisons  and  their  Antidotes,  Abbreviations 
Used  in  Prescriptions,  etc.  By  ELIAS  LONGLEY.  Cloth,  .75  ;  Tucks  and  Pocket,  $1.00 

Macalister's  Human  Anatomy.      816  Illustrations. 
Systematic  and  Topographical,    including  the  Embryology,    Histology,    and    Mor- 
phology of  Man.     With  Special  Reference  to  the  Requirements  of  Practical  Surgery 
and  Medicine.     By  ALEX.  MACALISTER,  M.D.,  F.R.S.,  Professor  of  Anatomy  in  the 
University  of  Cambridge.     816  Illustrations.     Octavo.     Cloth,  $5.00;  Leather,  $6.00 

McBride.  Diseases  of  the  Throat,  Nose,  and  Ear. 
A  Clinical  Manual  for  Students  and  Practitioners.  By  P.  McBRiDE,  M.D.,  F.R.C.P. 
(Edin.),  Surgeon  to  the  Ear  and  Throat  Department  of  the  Royal  Infirmary  ;  Lec- 
turer on  Diseases  of  Throat  and  Ear,  Edinburgh  School  of  Medicine,  etc.  With 
Colored  Illustrations  from  Original  Drawings.  Third  Edition.  Thoroughly  Revised 
and  Enlarged.  Octavo.  Handsome  Cloth,  Gilt  Top,  $7.00 

McCook.     American  Spiders  and  Their  Spinning  Work. 
A  Natural  History  of  the  Orbweaving  Spiders  of  the  United  States.     By  HENRY  C. 
McCooK,  D.D.,  Vice-President  of  the  Academy  of  Natural  Sciences  of  Philadelphia  ; 
Member  Entomological  Society  ;  Author  of  "The  Agricultural  Ants  of  Texas,"  etc. 
Three  volumes.     Handsomely  Illustrated.  Cloth,  $40.00 


MEDICAL   AND   SCIENTIFIC  PUBLICATIONS.  27 


Macready.     A  Treatise  on  Ruptures. 

By  JONATHAN  F.  C.  H.  MACREADY,  F.R.C.S.,  Surgeon  to  the  Great  Northern  Central 
Hospital ;  to  the  City  of  London  Hospital  for  Diseases  of  the  Chest,  etc.  24  Full- 
page  Plates  and  Wood  Engravings.  Octavo.  Cloth,  $6.00 

McFarland.     Prophylaxis — Personal  Hygiene — Nursing  and  Care  of 

the  Sick. 
See  COHEN,  Physiologic  Therapeutics,  page  10. 

McMurrich.     A  Manual  of  Embryology. 

By  J.  PLAYFAIR  McMuRRiCH,  A.M.,  PH.D.,  Professor  of  Anatomy,  Medical  Depart- 
ment of  the  University  of  Michigan,  Ann  Arbor.  276  Illustrations.  Just  Ready. 

Makins.     Surgical  Experiences  in  South  Africa,  1899—1900. 

Being  mainly  a  Clinical  Study  of  the  Effects  of  Injuries  Produced  by  Bullets  of  Small 
Calibre.  By  GEORGE  HENRY  MAKINS,  F.R.C.S.,  Surgeon  to  St.  Thomas's  Hospital, 
London  ;  Joint  Lecturer  on  Surgery  in  the  Medical  School  of  St.  Thomas's  Hospital  ; 
and  late  one  of  the  Consulting  Surgeons  to  the  South  African  Field  Force.  With  25 
Plates  and  96  other  Illustrations.  Octavo.  Cloth,  $4.00 

Mann.     Forensic  Medicine  and  Toxicology. 

By  J.  DIXON  MANN,  M.D.,  F.R.C.P.,  Professor  of  Medical  Jurisprudence  and  Toxi- 
cology in  Owens  College,  Manchester  ;  Examiner  in  Forensic  Medicine  in  University 
of  London,  etc.  Illustrated.  Octavo.  Cloth,  $6.50 

Mann's  Manual  of  Psychological  Medicine 

and  Allied  Nervous  Diseases.  Their  Diagnosis,  Pathology,  Prognosis,  and  Treat- 
ment, including  their  Medico-Legal  Aspects.  With  Chapter  on  Expert  Testimony  and 
an  Abstract  of  the  Laws  Relating  to  the  Insane  in  all  the  States  of  the  Union.  By 
EDWARD  C.  MANN,  M.D.  With  Illustrations.  Octavo.  Cloth,  $3.00 

Marshall's  Physiological  Diagrams,  Life  Size,  Colored. 

Eleven  Life-size  Diagrams  (each  7  feet  by  3  feet  7  inches).  Designed  for  Demon- 
stration before  the  Class.  By  JOHN  MARSHALL,  F.R.S.,  F.R.C.S.,  Professor  of 
Anatomy  to  the  Royal  Academy  ;  Professor  of  Surgery,  University  College,  London, 
etc.  In  Sheets,  $40.00  ;  Backed  with  Muslin  and  Mounted  on  Rollers,  $60.00 

Ditto,  Spring  Rollers,  in  Handsome  Walnut  Map  Case,  $100.00 
Single  Plates,  Sheets,  $5.00  ;  Mounted,  $7.50  ;  Explanatory  Key,  50  cents. 

Purchaser  must  pay  freight  charges. 

No.  i — The  Skeleton  and  Ligaments.  No.  2 — The  Muscles  and  Joints,  with  Ani- 
mal Mechanics.  No.  3 — The  Viscera  in  Position.  No.  4 — The  Heart  and  Principal 
Blood-vessels.  No.  5 — The  Lymphatics.  No.  6 — The  Digestive  Organs.  No.  7 — The 
Brain  and  Nerves.  Nos.  8  and  9 — The  Organs  of  the  Senses.  Nos.  10  and  11 — The 
Microscopic  Structure  of  the  Textures  and  Organs.  (Send  for  Special  Circular.} 

Maxwell.     Terminologia  Medica  Polyglotta. 

By  DR.  THEODORE  MAXWELL.     Octavo.  Cloth,  $3.00 

The  object  of  this  work  is  to  assist  the  medical  men  of  any  nationality  in  reading  medical 
literature  written  in  a  language  not  their  own.  Each  term  is  usually  given  in  seven  languages, 
viz.  :  English,  French,  German,  Italian,  Spanish,  Russian,  and  Latin. 

Maylard.-    The  Surgery  of  the  Alimentary  Canal. 
By  ALFRED  ERNEST  MAYLARD,  M.B.,  B.S.,  Senior  Surgeon  to  the  Victoria  Infirmary, 
Glasgow.     Second  Edition.     97  Illustrations.     Octavo.  Cloth,  $3.00 

Mays'  Theine  in  the  Treatment  of  Neuralgia. 
By  THOMAS  J.  MAYS,  M.D.      i6mo.  ^  bound,  .50 


28  P.   BLAKISTON'S  SON  6-    CO.1 S 

Memminger.     Diagnosis  by  the  Urine. 

The  Practical  Examination  of  Urine,  with  Special  Reference  to  Diagnosis.  By 
ALLARD  MEMMINGER,  M.D.,  Professor  of  Chemistry  and  Hygiene  ;  Clinical  Professor 
of  Urinary  Diagnosis  in  the  Medical  College  of  the  State  of  South  Carolina  ;  Visiting 
Physician  in  the  City  Hospital  of  Charleston,  etc.  Second  Edition,  Enlarged  and 
Revised.  24  Illustrations.  I2mo.  Cloth,  $1.00 

Minot.     Embryology. 

A  Laboratory  Text-Book  of  Embryology.  By  CHARLES  S.  MINOT,  S.D.,  LL.D.,  Pro- 
fessor of  Histology  and  Human  Embryology,  Harvard  University  Medical  School. 
Illustrated.  Nearly  Ready. 

Montgomery.     A  Text-Book  of  Practical  Gynecology. 
By  EDWARD  E.  MONTGOMERY,  M.D.,  Professor  of  Gynecology  in  Jefferson  Medical 
College,  Philadelphia  ;  Gynecologist  to  the  Jefferson  and  St.  Joseph's  Hospitals,  etc. 
527  Illustrations,  many  of  which    are  from  original  sources.     800  pages.     Octavo. 

Cloth,  $5.00  ;  Leather,  $6.00 

%*  This  is  a  systematic  modern  treatise  on  Diseases  of  Women.  The  author's 
aim  has  been  to  produce  a  book  that  will  be  thorougji  and  practical  in  every  particular. 
The  illustrations,  nearly  all  of  which  are  from  original  sources,  have  for  the  most  part 
been  drawn  by  special  artists. 

"  The  author  has  a  clear  conception  of  his  subject ;  this,  with  his  manner  of  treatment,  intro- 
duces the  reader  to  questions  otherwise  intricate  in  such  a  manner  as  to  make  them  easily  compre- 
hended. His  introduction,  together  with  his  comments  on  diagnosis  and  examination  of  the 
patient  are  delightfully  clear  and  instructive.  Therapeutics,  local  and  systematic,  are  clearly  and 
intelligently  discussed." — Brooklyn  Mfdical  Journal. 

Morris.     Text-Book  of  Anatomy.     Third  Edition.      846   Illustra- 
tions, 267  in  Colors. 

A  Complete  Text-Book.  Edited  by  HENRY  MORRIS,  F.R.C.S.,  Surgeon  to,  and  Lec- 
turer on  Anatomy  at,  Middlesex  Hospital,  assisted  by  J.  BLAND  SUTTON,  F.R.C.S., 

J.   H.   DAVIES-COLLEY,   F.R.C.S.,  WM.  J.  WALSHAM,   F.R.C.S.,   H.   ST.    JOHN    BROOKS, 

M.D.,  R.  MARCUS  GUNN,  F.R.C.S.,  ARTHUR  HENSMAN,  F.R.C.S.,  FREDERICK  TREVES, 
F.R.C.S.,  WILLIAM  ANDERSON,  F.R.C.S.,  ARTHUR  ROBINSON,  M.D.,  M.R.C.S.,  and 
PROF.  W.  H.  A.  JACOBSON.  One  Handsome  Octavo  Volume,  with  846  Illustrations, 
of  which  267  are  printed  in  Colors.  Thumb  Index  and  Colored  Illustrations  in  all 
Copies.  Cloth,  $6.00  ;  Leather,  $7.00  ;  Half  Russia,  #8.00 

"  Of  all  the  text-books  of  moderate  size  on  human  anatomy  in  the  English  language,  Morris 
is  undoubtedly  the  most  up-to-date  and  accurate.  .  .  .  For  the  student,  the  surgeon,  or  for  the 
general  practitioner  who  desires  to  review  his  anatomy,  Morris  is  decidedly  the  book  to  buy." — 
The  Philadelphia  Medical  Journal. 

*q*  MORRIS'  ANATOMY  is  now  the  recognized  standard  text-book  in  a  large  number 
of  medical  schools  throughout  the  United  States,  England,  and  Canada.  It  is  in  many 
respects  the  best  book  for  students'  use,  and  in  its  present  edition  is  the  latest  and  best 
illustrated  of  all  books  on  anatomy.  The  revisions  have  been  carefully  made  and 
edited,  several  sections  having  been  almost  entirely  rewritten,  old  illustrations  replaced 
and  new  ones  added,  a  larger  number  being  printed  in  colors. 

Renal  Surgery. 

With  Special  Reference  to  Stone  in  the  Kidney  and  Ureter,  and  to  the  Surgical 
Treatment  of  Calculous  Anuria,  together  with  a  Critical  Examination  of  Sub- 
parietal  Injuries  of  the  Ureter.  Illustrated.  8vo.  Cloth,  $2.00 

Mitchell  and  Gulick.      Mechanotherapy. 
See  COHEN,  Physiologic  Therapeutics,  page  10. 


MEDICAL  AND   SCIENTIFIC  PUBLICATIONS.  29 

Morton  on  Refraction  of  the  Eye. 

Its  Diagnosis  and  the  Correction  of  its  Errors.  With  Chapter  on  Keratoscopy  and 
Test  Types.  By  A.  MORTON,  M.B.  Sixth  Edition,  Revised.  Cloth,  $1.00 

Moullin.  Surgery.  Third  Edition,  by  Hamilton. 
A  Complete  Text-Book.  By  C.  W.  MANSELL  MOULLIN,  M.A.,  M.D.  (Oxon.),  F.R.C.S., 
Surgeon  and  Lecturer  on  Physiology  to  the  London  Hospital ;  formerly  Radcliffe 
Traveling  Fellow  and  Fellow  of  Pembroke  College,  Oxford.  Third  American 
Edition,  Revised  and  Edited  by  the  late  JOHN  B.  HAMILTON,  M.D.,  LL.D.,  Professor 
of  the  Principles  of  Surgery  and  Clinical  Surgery,  Rush  Medical  College,  Chicago  ; 
Professor  of  Surgery,  Chicago  Polyclinic  ;  Surgeon,  formerly  Supervising  Surgeon- 
General,  U.  S.  Marine  Hospital  Service  ;  Surgeon  to  Presbyterian  Hospital.  600 
Illustrations,  over  200  of  which  are  original,  and  many  of  which  are  printed  in 
Colors.  Octavo.  1250  pages.  Cloth,  $6.00;  Leather,  $7.00;  Half  Russia,  $8.00 

Enlargement  of  the  Prostate. 

Its   Treatment   and    Radical    Cure.     Illustrated.     Second    Edition,    Enlarged. 
Octavo.  Cloth,  $1.75 

Inflammation  of  the  Bladder  and  Urinary  Fever. 

Octavo.  Cloth,  $1.50 

Murray.     Rough  Notes  on  Remedies. 

By  WM.  MURRAY,  M.D.,  F.R.C.P.  (Lond.),  Consulting  Physician  Newcastle-on-Tyne 
Hospital  for  Sick  Children.  Fourth  Edition,  Enlarged.  Crown  8vo.  Cloth,  $1.25 

Muter.     Practical  and  Analytical  Chemistry. 

By  JOHN  MUTER,  F.R.S.,  F.C.S.,  etc.  Second  American  from  the  Eighth  English 
Edition.  Revised  to  meet  the  Requirements  of  American  Medical  and  Pharma- 
ceutical Colleges.  56  Illustrations.  Cloth,  $1.25 

New  Sydenham  Society  Publications. 

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Per  annum,  $8.00 

Notter.  The  Theory  and  Practice  of  Hygiene.  Second  Edition. 
A  Complete  Treatise  by  J.  LANE  NOTTER,  M.A.,  M.D.,  F.C.S.,  Fellow  and  Member 
of  Council  of  the  Sanitary  Institute  of  Great  Britain  ;  Professor  of  Hygiene,  Army 
Medical  School;  Examiner  in  Hygiene,  University  of  Cambridge,  etc.;  and  W.  H. 
HORROCKS,  M.D.,  B.  Sc.  (Lond.),  Assistant  Professor  of  Hygiene,  Army  Medical 
School,  Netley.  Illustrated  by  15  Lithographic  Plates  and  138  other  Illustrations, 
and  including  many  Useful  Tables.  Second  Edition,  Carefully  Revised.  Octavo. 
1085  pages.  Cloth,  $7.00 

Oertel.     Medical  Microscopy. 

A  Guide  to  Diagnosis,  Elementary  Laboratory  Methods,  and  Microscopic  Technic. 
By  T.  E.  OERTEL,  M.D.,  Professor  of  Pathology  and  Clinical  Microscopy,  Medical 
Department,  University  of  Georgia.  I2mo.  120  Illustrations.  Nearly  Ready. 

Oettel.     Practical  Exercises  in  Electro-Chemistry. 

By  DR.  FELIX  OETTEL.  Authorized  Translation  by  EDGAR  F.  SMITH,  M.A.,  Professor 
of  Chemistry,  University  of  Pennsylvania.  Illustrated.  Cloth,  .75 

Introduction  to  Electro-Chemical  Experiments. 

Illustrated.     By  same  Author  and  Translator.  Cloth,  .75 


30  P.   BLAKISTON'S  SON  &*    CO.' S 

Ohlemann.     Ocular  Therapeutics  for  Physicians  and  Students. 

By  M.  OHLEMANN,  M.D.,  late  Physician  in  the  Ophthalmological  Clinical  Institute, 
Royal  Prussian  University  of  Berlin,  etc.  Translated  and  Edited  by  CHARLES  A. 
OLIVER,  A.M.,  M.D.,  Attending  Surgeon  to  the  Wills  Eye  Hospital ;  Ophthalmic  Surgeon 
to  the  Philadelphia  and  to  the  Presbyterian  Hospitals.  I2mo.  Cloth,  $1.75 

Ormerod.     Diseases  of  Nervous  System. 

By  J.  A.  ORMEROD,  M.D.  (Oxon.),  F.R.C.P.,  Physician  to  National  Hospital  for  the 
Paralyzed  and  Epileptic,  London.  66  Wood  Engravings.  i2mo.  Cloth,  $1.00 

Osgood.     The  Winter  and  Its  Dangers. 

By  HAMILTON  OSGOOD,  M.D.  Cloth,  .40 

Ostrom.     Massage  and  the  Original  Swedish  Movements. 

Their  Application  to  Various  Diseases  of  the  Body.  A  Manual  for  Students,  Nurses, 
and  Physicians.  By  KURRE  W.  OSTROM,  from  the  Royal  University  of  Upsala, 
Sweden,  Formerly  Instructor  in  Massage  and  Swedish  Movements  in  the  Hospital  of 
the  University  of  Pennsylvania  and  in  the  Philadelphia  Polyclinic  and  College  for 
Graduates  in  Medicine,  etc.  Fifth  Edition,  Enlarged.  1 1 5  Illustrations,  many  of 
which  were  drawn  especially  for  this  purpose.  I2mo.  Just  Ready.  Cloth,  $1.00 

Packard's  Sea  Air  and  Sea  Bathing. 

By  JOHN  H.  PACKARD,  M.D.  Cloth,  .40 

Parkes.     Hygiene  and  Public  Health. 

A  Practical  Manual.  By  Louis  C.  PARKES,  M.D.,  D.P.H.  (Lond.  Univ.),  Lecturer 
on  Public  Health  at  St.  George's  Hospital ;  Medical  Officer  of  Health  and  Public 
Analyst,  Borough  of  Chelsea,  London,  etc.;  and  HENRY  KENWOOD,  M.B.,  F.C.S., 
Assistant  Professor  of  Public  Health,  University  College,  London,  etc.  Sixth  Edition, 
Enlarged  and  Revised.  85  Illustrations.  I2mo.  Just  Ready.  Cloth,  $3.00 

"  The  style  is  good;  dry  facts,  laws,  and  statistics  are  put  in  such  a  way  that  the  reader  does 
not  tire  of  them  and  yet  finds  them  easy  to  lemember." — University  Medical  Magazine. 

The  Elements  of  Health. 

An  Introduction  to  the  Study  of  Hygiene.     Illustrated.  Cloth,  $1.25 

Parsons.     Elementary  Ophthalmic  Optics. 

By  J.  HERBERT  PARSONS,  M.B.,  M.R.C.S.,  Clinical  Assistant,  Royal  London  Ophthal- 
mic Hospital.  With  Diagrammatic  Illustrations.  Just  Ready.  Cloth,  $2.00 

Pershing.     The  Diagnosis  of  Nervous  and  Mental  Diseases. 

By  HOWELL  T.  PERSHING,  M.D.,  Professor  of  Nervous  and  Mental  Diseases  in  the 
University  of  Denver;  Neurologist  to  St.  Luke's  Hospital;  Consultant  in  Nervous 
and  Mental  Diseases  to  the  Arapahoe  County  Hospital  ;  Member  of  the  American 
Neurological  Association.  With  colored  and  other  Illustrations.  Cloth,  $1.25 

Phillips.     Spectacles  and  Eyeglasses. 

Their  Prescription  and  Adjustment.  By  R.  J.  PHILLIPS,  M.D.,  Instructor  in  Diseases 
of  the  Eye,  Philadelphia  Polyclinic  ;  Ophthalmic  Surgeon,  Presbyterian  Hospital. 
Second  Edition,  Revised  and  Enlarged.  49  Illustrations.  I2mo.  Cloth,  $1.00 


MEDICAL   AND   SCIENTIFIC  PUBLICATIONS.  31 

The  Physician's  Visiting  List. 

Published  Annually.     Fifty-Second  Year  (1903)  of  its  Publication. 

Hereafter  all  styles  will  contain  the  interleaf  or  special  memoranda  page,  except 
the  Monthly  Edition,  and  the  sizes  for  75  and  100  Patients  will  come  in  two  volumes 
only. 

REGULAR    EDITION. 

For  25  Patients  Weekly.  Tucks,  pocket  and  pencil,  Gilt  Edges,  $1.00 

5°         "  " 


75  "    -ol,  " 

,         f  Jan.  to  Tune  I  , 
2vols"     I  July  to  Dec.  I  2'25 

Perpetual  Edition, 

without  Dates  and  with  Special  Memorandum  Pages. 

For  25  Patients,  Interleaved,  Tucks,  Pocket,  and  Pencil,  $1.25 
For  50  Patients,  Interleaved,  Tucks,  Pocket,  and  Pencil,  $1.50 

Monthly  Edition,  without  Dates. 

Can  be  commenced  at  any  time  and  used  until  full.     Requires  only  one  writing 
of  patient's  name  for  the  whole  month. 
Plain  binding,  without  Flap  or  Pencil,  .75  ;  Leather  cover,  Pocket  and  Pencil,  $1.00 

Extra  Pencils 

will  be  sent,  postpaid,  for  25  cents  per  half  dozen. 

This  list  combines  the  several  essential  qualities  of  strength,   compactness, 


durability,  and  convenience.  It  is  made  in  all  sizes  and  styles  to  meet  the  wants  of  all 
physicians.  It  is  not  an  elaborate,  complicated  system  of  keeping  accounts,  but  a 
plain,  simple  record,  that  may  be  kept  with  the  least  expenditure  of  time  and  trouble — 
hence  its  popularity.  A  special  circular,  descriptive  of  contents,  will  be  sent  upon 
application. 

Potter.     A  Handbook  of  Materia  Medica,  Pharmacy,  and  Thera- 
peutics.    Ninth  Edition,  Enlarged. 

Including  the  Action  of  Medicines,  Special  Therapeutics  of  Disease,  Official  and 
Practical  Pharmacy,  and  Minute  Directions  for  Prescription  Writing,  etc.  Including 
over  650  Prescriptions  and  Formulae.  By  SAMUEL  O.  L.  POTTER,  M.A.,  M.D.,  M.R.C.P. 
(Lond.),  formerly  Professor  of  the  Principles  and  Practice  of  Medicine,  Cooper  Medical 
College,  San  Francisco  ;  Major  and  Brigade  Surgeon,  U.  S.  Vol.  Ninth  Edition, 
Revised  and  Enlarged.  8vo.  Just  Ready. 
With  Thumb  Index  in  each  copy.  Cloth,  $5.00  ;  Leather,  $6.00  ;  Half  Russia,  $7.00 

Compend  of  Anatomy,  including  Visceral  Anatomy. 

Sixth  Edition,   Revised  and  greatly  Enlarged.     With    16  Lithographed  Plates 
and  117  other  Illustrations.     Being  No.  i  ?  Quiz-Compend  ?  Series. 

Cloth,  .80  ;  Interleaved  for  Taking  Notes,  $1.00 

Compend   of    Materia   Medica,   Therapeutics,  and    Prescription 
Writing. 

With  Special  Reference  to  the  Physiological  Action  of  Drugs.  '  Sixth  Revised  and 
Improved  Edition,  with  Index.     Being  No.  6  f  Quiz -Compend  f  Series. 

Cloth,  .80;  Interleaved  for  Taking  Notes,  $1.00 


32  P.   BLAKISTON'S  SON  &>   CO.' S 

Potter.     Speech  and  Its  Defects. 

Considered  Physiologically,  Pathologically,  and  Remedially  ;  being  the  Lea  Prize 
Thesis  of  Jefferson  Medical  College,  1882.  Revised  and  Corrected.  Cloth,  $1.00 

Power.     Surgical  Diseases  of  Children 

and  their  Treatment  by  Modern  Methods.  By  D'AncY  POWER,  M.A.,  F.R.C.S. 
(Eng.),  Demonstrator  of  Operative  Surgery,  St.  Bartholomew's  Hospital;  Surgeon 
to  the  Victoria  Hospital  for  Children.  Illustrated.  I2mo.  Cloth,  $2.50 

Preston.     Hysteria  and  Certain  Allied  Conditions. 

Their  Nature  and  Treatment.  With  Special  Reference  to  the  Application  of  the  Rest 
Cure,  Massage,  Electrotherapy,  Hypnotism,  etc.  By  GEORGE  J.  PRESTON,  M.D., 
Professor  of  Diseases  of  the  Nervous  System,  College  of  Physicians  and  Surgeons, 
Baltimore  ;  Visiting  Physician  to  the  City  Hospital ;  Consulting  Neurologist  to  Bay 
View  Asylum  and  the  Hebrew  Hospital ;  Member  American  Neurological  Associa- 
tion, etc.  Illustrated.  I2mo.  Cloth,  $2.00 

Pritchard.     Handbook  of  Diseases  of  the  Ear. 

By  URBAN  PRITCHARD,  M.D.,  F.R.C.S.,  Professor  of  Aural  Surgery,  King's  College, 
London;  Aural  Surgeon  to  King's  College  Hospital;  Senior  Surgeon  to  the  Royal 
Ear  Hospital,  etc.  Third  Edition.  Many  Illustrations  and  Formulae.  Cloth,  $1.50 

Proctor's  Practical  Pharmacy. 

Lectures  on  Practical  Pharmacy.  By  BARNARD  S.  PROCTOR.  Third  Edition,  Re- 
vised. With  Elaborate  Tables  of  Chemical  Solubilities,  etc.  Illustrated.  Cloth,  $3.00 

Reese's  Medical  Jurisprudence  and  Toxicology. 

A  Text-Book  for  Medical  and  Legal  Practitioners  and  Students.  By  JOHN  J.  REESE, 
M.D.,  Editor  of  "Taylor's  Jurisprudence,"  formerly  Professor  of  the  Principles  and 
Practice  of  Medical  Jurisprudence,  including  Toxicology,  in  the  University  of  Pennsyl- 
vania Medical  Department.  Fifth  Edition,  Revised  and  Edited  by  HENRY  LEFFMANN, 
M.D.,  Pathological  Chemist,  Jefferson  Medical  College  Hospital ;  Chemist,  State  Board 
of  Health  ;  Professor  of  Chemistry,  Woman's  Medical  College  of  Pennsylvania,  etc. 
I2mo.  645  pages.  Cloth,  $3.00;  Leather,  $3.50 

"  To  the  student  of  medical  jurisprudence  and  toxicology  it  is  invaluable,  as  it  is  concise, 
clear,  and  thorough  in  every  respect." — The  American  Journal  of  the  Medical  Sciences. 

Reeves.      Medical  Microscopy. 

Including  Chapters  on  Bacteriology,  Neoplasms,  Urinary  Examination,  etc.  By 
JAMES  E.  REEVES,  M.D.,  ex-President  American  Public  Health  Association,  etc. 
Numerous  Illustrations,  some  of  which  are  printed  in  Colors.  i2mo.  Cloth,  $2.50 

Regis.     Mental  Medicine. 

A  Practical  Manual.  By  DR.  E.  REGIS,  formerly  Chief  of  Clinique  of  Mental  Dis- 
eases, Faculty  of  Medicine  of  Paris.  Authorized  Translation  by  H.  M.  BANNISTER, 
M.D.,  late  Senior  Assistant  Physician,  Illinois  Eastern  Hospital  for  the  Insane,  etc. 
With  an  Introduction  by  the  Author.  I2mo.  Cloth,  $2.00 

Richardson.      Long  Life 

and  How  to  Reach  It.  By  J.  G.  RICHARDSON,  formerly  Professor  ot  Hygiene,  Uni- 
versity of  Pennsylvania.  .  Cloth,  .40 

Rockwood.     Chemical  Analysis. 

Introduction  to  Chemical  Analysis  for  Students  of  Medicine,  Pharmacy,  and  Dentistry. 
By  ELBERT  W."  ROCKWOOD,  B.S.,  M.D.,  Professor  of  Chemistry,  Toxicology,  and 
Metallurgy  in  the  Colleges  of  Medicine,  Dentistry,  and  Pharmacy,  University  of  Iowa, 
Iowa  City.  Illustrated.  Cloth,  $1.50 


MEDICAL   AND   SCIENTIFIC  PUBLICATIONS.  33 

Richardson's  Mechanical  Dentistry. 

A  Practical  Treatise  on  Mechanical  Dentistry.  By  JOSEPH  RICHARDSON,  D.D.S. 
Seventh  Edition,  Thoroughly  Revised  and  in  many  parts  Rewritten  by  GEO.  W. 
WARREN,  A.M.,  D.D.S.,  Professor  of  Clinical  Dentistry  and  Oral  Surgery;  Chief  of 
the  Clinical  Staff,  Pennsylvania  College  of  Dental  Surgery,  Philadelphia.  With  691 
Illustrations.  Octavo.  675  pages.  Cloth,  #5.00 ;  Leather,  $6.00 

Richter's  Inorganic  Chemistry. 

A  Text-Book  for  Students.  By  PROF.  VICTOR  VON  RICHTER,  University  of  Breslau. 
Fifth  American  from  Tenth  German  Edition  by  PROF.  H.  KLINGER,  University  of 
Konigsberg.  Authorized  Translation  by  EDGAR  F.  SMITH,  M.A.,  PH.D.,  SC.D., 
Professor  of  Chemistry,  University  of  Pennsylvania  ;  Member  of  the  Chemical  Society 
of  Berlin,  etc.  With  many  Illustrations  and  a  Colored  Plate.  I2mo.  Cloth,  $1.75 

Organic  Chemistry. 

The  Chemistry  of  the  Carbon  Compounds.  Third  American  Edition,  Translated 
from  PROF.  ANSCHUTZ'S  Eighth  German  Edition  by  EDGAR  F.  SMITH,  M.A., 
PH.D.,  SC.D.,  Professor  of  Chemistry,  University  of  Pennsylvania.  Revised 
and  Enlarged.  Illustrated.  I2mo.  Two  volumes. 

Vol.    I.     Aliphatic  Series.     625  pages.  Cloth,  $3.00 

Vol.  II.     Carbocyclic  and  Heterocyclic  Series.     671  pages.  Cloth,  $3.00 

Roberts.     Gynecological  Pathology. 

Gynecological  Pathology.    By  CHARLES  HURBERT  ROBERTS,  M.D.,  F.R.C.S.,  M.R.C.P., 

Physician  Queen  Charlotte's  Lying-in  Hospital  and  to  the  Samaritan  Hospital  for 

Women  ;  Demonstrator  of  Practical  Midwifery  and  Diseases  of  Women,  and  House 

Surgeon     St.     Bartholomew's     Hospital,     London.       Elaborately    Illustrated    with 

127    Full-Page    Plates    containing    151    Figures,    several   being   printed   in  Colors. 

Octavo.  Extra  Cloth,  Gilt  Top,  $6.00 

"This  very  attractive  volume  will  be  appreciated  by  all  who  teach  gynecology  or  who  deal 

practically  with  diseases  of  the  female  reproductive  organs." — Edinburgh  Medical  Journal. 

"  This  is  a  splendid  volume,  worthy  the  careful  study  of  every  physician  who  seeks  to  under- 
stand the  diseased  conditions  he  so  often  meets  with  in  his  female  patients." — Annals  of  Gynecology 
and  Pediatry. 

Robinson.     Latin  Grammar  of  Pharmacy  and  Medicine. 
By  D.  H.  ROBINSON,  PH.D.,  Professor  of  Latin  Language  and  Literature,  University 
of  Kansas.     Introduction    by    L.    E.    SAYRE,    PH.G.,    Professor   of  Pharmacy   and 
Dean  of  the  Department   of  Pharmacy  in  University  of  Kansas.     Third  Edition, 
Revised  with  the  help  of  PROF.   L.   E.  SAYRE,  of  University  of  Kansas,  and  DR. 
CHARLES  RICE,  of  the  College  of  Pharmacy  of  the  City  of  New  York.     Cloth,  $1.75 
"  This  method  of  preparing  medical  students  and  pharmacists  for  a  practical  use  of  the  lan- 
guage is  in  every  way  to  be  commended.     .     .     .     Pharmacists  should  know  enough  to  read  pre- 
scriptions readily  and  understandingly." — Johns  Hopkins  Hospital  Bulletin. 

Rosenau.     Disinfection  and  Disinfectants. 

A  Practical  Guide  for  Sanitarians,  Health  and  Quarantine  Officers.  By  M.  J.  ROSE- 
NAU, M.D.,  Director  of  the  Hygienic  Laboratory  and  Passed  Assistant  Surgeon,  U.  S. 
Marine  Hospital  Service,  Washington,  D.  C.  Illustrated.  Just  Ready.  Cloth,  $2.00 

Sayre.     Organic  Materia  Medica  and  Pharmacognosy. 

An  Introduction  to  the  Study  of  the  Vegetable  Kingdom  and  the  Vegetable  and 
Animal  Drugs.  Comprising  the  Botanical  and  Physical  Characteristics,  Source, 
Constituents,  Pharmacopceial  Preparations  ;  Insects  Injurious  to  Drugs,  and  Phar- 
macal  Botany.  By  L.  E.  SAYRE,  B.S.,  PH.M.,  Dean  of  the  School  of  Pharmacy  and 
Professor  of  Materia  Medica  and  Pharmacy  in  the  University  of  Kansas  ;  Member 
Committee  of  Revision  of  the  United  States  Pharmacopoeia,  etc.  With  Sections  on 
Histology  and  Microtechnique  by  WILLIAM  C.  STEVENS,  Professor  of  Botany  in  the 
University  of  Kansas.  Second  Edition,  Revised  and  Enlarged.  With  374  Illustra- 
tions, the  majority  of  which  are  from  Original  Drawings.  8vo.  Cloth,  $4.50 


34  P.    BLAKISTON'S  SON  6-    CO.' S 

Schamberg.     Compend  of  Diseases  of  the  Skin. 

By  JAY  F.  SCHAMBERG,  Professor  of  Diseases  of  the  Skin,  Philadelphia  Polyclinic  ; 
Fellow  of  the  College  of  Physicians  of  Philadelphia  ;  Quiz-Master  at  University  of 
Pennsylvania.  Second  Edition,  Revised  and  Enlarged.  105  Illustrations,  f  Quiz- 
Compend?  Series,  No.  16.  Cloth,  .80;  Interleaved,  $1.00 

Schofield.     The  Force  of  Mind, 

or  The  Mental  Factor  in  Medicine.  By  A.  T.  SCHOFIELD,  M.D.,  M.R.C.S.  Just 
Ready.  Cloth,  $2.00 

Schreiner.     Diet  List. 

Arranged  in  the  Form  of  a  Chart  on  which  Articles  of  Diet  can  be  Indicated  for  any 
Disease.  By  E.  R.  SCHREINER,  M.D.,  Assistant  Demonstrator  of  Physiology,  Uni- 
versity of  Pennsylvania.  Put  up  in  Pads  of  50  with  Pamphlet  of  Specimen  Dietaries. 

Per  Pad,  .75 

Scott.     The  Urine  :   Its  Chemical  and  Microscopical  Examination. 

By  LINDLEY  MARCROFT  SCOTT,  M.A.,  M.D.,  etc.  With  41  Colored  Plates  and  other 
Illustrations.  Quarto.  Cloth,  $5.00 

Scoville.     The  Art  of  Compounding.     Second  Edition. 

A  Text-Book  for  Students  and  a  Reference  Book  for  Pharmacists.  By  WILBUR  L. 
SCOVILLE,  PH.G.,  Professor  of  Applied  Pharmacy  and  Director  of  the  Pharmaceutical 
Laboratory  in  the  Massachusetts  College  of  Pharmacy.  Second  Edition,  Enlarged 
and  Improved.  Cloth,  $2.50;  Sheep,  $3.50;  Half  Russia,  $4.50 

Self-Examination  for  Medical-  Students. 

3500  Questions  on  Medical  Subjects,  with  the  proper  References  to  Standard  Books 
in  which  replies  may  be  found,  and  including  Complete  Sets  of  Questions  from  two 
recent  State  Board  Examinations  of  Pennsylvania,  Illinois,  and  New  York.  641110. 

Paper,  10  cents. 

Smith.     Abdominal  Surgery.     Sixth  Edition. 

Being  a  Systematic  Description  of  all  the  Principal  Operations.  By  J.  GREIG  SMITH, 
M.A.,  F.R.S.E.,  Surgeon  to  British  Royal  Infirmary.  224  Illustrations.  Sixth  Edition, 
Enlarged  and  Thoroughly  Revised  by  JAMES  SWAIN,  M.D.  (Lond.),  F.R.C.S.,  Pro- 
fessor of  Surgery,  University  College,  Bristol,  etc.  Two  vols.  8vo.  Cloth,  $10.00 

Smith.     Electro-Chemical  Analysis. 

By  EDGAR  F.  SMITH,  M.A.,  PH.D.,  SC.D.,  Professor  of  Chemistry,  University  of 
Pennsylvania.  Second  Edition,  Revised  and  Enlarged.  27  Illustrations.  I2mo. 

Cloth,  $1.25 
*^*  This  book  has  been  translated  and  published  in  both  Germany  and  France. 

Smith  and  Keller.     Experiments. 

Arranged  for  Students  in  General  Chemistry.  By  EDGAR  F.  SMITH,  M.A.,  PH.D., 
SC.D.,  Professor  of  Chemistry,  University  of  Pennsylvania,  and  DR.  H.  F.  KELLER, 
Professor  of  Chemistry,  Philadelphia  High  School.  Fourth  Revised  Edition.  8vo. 
Illustrated.  Cloth,  .60 

Smith.     Dental  Metallurgy. 

A  Manual.  By  ERNEST  A.  SMITH,  F.C.S.,  Assistant  Instructor  in  Metallurgy,  Royal 
College  of  Science,  London.  Illustrated.  Second  Edition.  In  Press. 

Smith.     Wasting  Diseases  of  Infants  and  Children. 

By  EUSTACE  SMITH,  M.D.,  F.R.C.P.,  Physician  to  the  East  London  Hospital  for 
Children,  etc.  Sixth  Edition,  Revised.  Cloth,  $2.00 


MEDICAL  AND   SCIENTIFIC  PUBLICATIONS.  35 

Starling.     Elements  of  Human  Physiology. 

By  ERNEST  H.  STARLING,  M.D.  (Lond.),  M.R.C.P.,  Joint  Lecturer  on  Physiology  at 
Guy's  Hospital,  London,  etc.  With  100  Illus.  121110.  437  pages.  Cloth,  $1.00 

Starr.     The  Digestive  Organs  in  Childhood. 

The  Diseases  of  the  Digestive  Organs  in  Infancy  and  Childhood.  By  Louis  STARR, 
M.D.,  late  Clinical  Professor  of  Diseases  of  Children  in  the  Hospital  of  the  University 
of  Pennsylvania ;  Physician  to  the  Children' s  Hospital,  Philadelphia.  Third 
Edition,  Revised  and  Enlarged.  Illustrated.  Octavo.  Just  Ready.  Cloth,  $3.00 

The  Hygiene  of  the  Nursery. 

Including  the  General  Regimen  and  Feeding  of  Infants  and  Children,  and  the 
Domestic  Management  of  the  Ordinary  Emergencies  of  Early  Life,  Massage,  etc. 
Sixth  Edition,  Enlarged.  25  Illustrations.  I2mo.  Cloth,  $1.00 

*#*  General  and  specific  rules  for  feeding  are  given,  and  Diet  Lists  from  the  first 
week  up  to  the  eighteenth  month,  with  various  recipes  for  artificial  foods,  peptonized 
milk,  etc.  Directions  for  the  sterilization  of  milk,  substitutes  for  milk,  preparation  of 
food  for  both  well  and  sick  children,  nutritious  enemata,  etc.,  and  the  general  manage- 
ment of  the  Nursery. 

Stearns.     Lectures  on  Mental  Diseases. 

By  HENRY  PUTNAM  STEARNS,  M.D.,  Physician-Superintendent  at  the  Hartford  Retreat ; 
Lecturer  on  Mental  Diseases  in  Yale  University.  With  a  Digest  of  Laws  of  the 
Various  States  Relating  to  Care  of  Insane.  Illustrated.  Cloth,  $2.75  ;  Sheep,  $3.25 

Steell.     The  Physical  Signs  of  Pulmonary  Disease. 

By  GRAHAM  STEEL,  M.D.,  F.R.C.P.,  Physician  to  the  Manchester  Royal  Infirmary  ; 
Lecturer  on  Clinical  Medicine  and  on  Diseases  of  the  Heart  at  Owens  College. 
Illustrated.  Cloth,  $1.25 

Stevenson  and  Murphy.     A  Treatise  on  Hygiene. 

By  Various  Authors.  Edited  by  THOMAS  STEVENSON,  M.D.,  F.R.C.P.,  Lecturer  on 
Chemistry  and  Medical  Jurisprudence  at  Guy's  Hospital,  London,  and  SHIRLEY  F. 
MURPHY,  Medical  Officer  of  Health  to  the  County  of  London.  In  three  octavo 
volumes. 

Vol.     I.     With  Plates  and  Wood  Engravings.     Octavo.  Cloth,  $6.00 

Vol.    II.     With  Plates  and  Wood  Engravings.     Octavo.  Cloth,  $6.00 

Vol.  III.     Sanitary  Law.     Octavo.  Cloth,  $5.00 

*•**  Special  Circular  upon  application. 

Stewart's  Compend  of  Pharmacy. 

Based  upon  "Remington's  Text-Book  of  Pharmacy."  By  F.  E.  STEWART,  M.D., 
PH.G.,  late  Quiz-Master  in  Chemistry  and  Theoretical  Pharmacy,  Philadelphia  College 
of  Pharmacy  ;  Lecturer  on  Pharmacology,  Jefferson  Medical  College.  Fifth  Edition. 
Complete  Tables  of  Metric  and  English  Weights  and  Measures.  ?  Quiz- Compend  ? 
Series.  Cloth,  .80  ;  Interleaved  for  the  Addition  of  Notes,  $1.00 

Stirling.     Outlines  of  Practical  Physiology. 

Including  Chemical  and  Experimental  Physiology,  with  Special  Reference  to  Practical 
Medicine.  By  W.  STIRLING,  M.D.,  SC.D.,  Professor  of  Physiology  and  Histology, 
Owens  College,  Victoria  University,  Manchester  ;  Examiner  in  Physiology,  Univer- 
sities of  Edinburgh  and  London.  Third  Edition.  289  Illustrations.  Cloth,  $2.00 

Outlines  of  Practical  Histology. 

368  Illustrations.  Second  Edition,  Revised  and  Enlarged.  With  new  Illustra- 
tions. I2mo.  Cloth,  $2.00 


36  P.  BLAKISTON'S  SON  &>   CO.'S 

Stohr.       Text-Book    of    Histology,    Including    the    Microscopical 
Technic.     379  Illustrations.     New  Edition. 

By  DR.  PHILIP  STOHR,  Professor  of  Anatomy  at  University  of  Wiirzburg.     Author- 
ized Translation  by  EMMA  L.  BILSTEIN,  M.D.,  formerly  Demonstrator  of  Histology, 
Woman's    Medical  College   of  Penna.     Edited,   with  Additions,  by   DR.  ALFRED 
SCHAPER,  Professor  of  Anatomy,  University  of  Breslau;  formerly  Demonstrator  of 
Histology,  Harvard  Medical  School,  Boston.    Fourth  American  based  upon  the  Ninth 
German  Edition,  Enlarged  and  Revised.     379  Illustrations.     Octavo.         Cloth,  #3.00 
"This  edition  of  an  already  well-known  student's  manual  requires  little  but  favorable  com- 
ment.    Its  other  editions  have  made  it  well  and  favorably  known,  and  this  one  only  makes  the 
work's  position  more  secure.     The  book  is  not  only  a  useful  one  for  the  student,  but  makes  a  very 
good  work  of  reference  for  its  subject,  and  is  thus  entitled  to  a  place  upon  the  shelves  of  the  prac- 
titioner."—  The  Medical  Record,  New  York, 

Sturgis.     Manual  of  Venereal  Diseases.     Seventh  Edition. 

By  F.  R.  STURGIS,  M.D.,  Sometime  Clinical  Professor  of  Venereal  Diseases  in  the 
Medical  Department  of  the  University  of  the  City  of  New  York  ;  formerly  one  of 
the  Visiting  Surgeons  to  Charity  Hospital,  Blackwells  Island,  Department  of  Vene- 
real Diseases  ;  Member  of  the  American  Association  of  Genito-Urinary  Surgeons,  etc. 
Seventh  Edition,  Revised  and  in  part  Rewritten  by  F.  R.  STURGIS,  M.D.,  and  FOLLEN 
CABOT,  M.  D. ,  Instructor  in  Genito-Urinary  and  Venereal  Diseases  in  the  Cornell  Uni- 
versity Medical  College  ;  Genito-Urinary  Out-Patient  Surgeon  to  Bellevue  Hospital ; 
Visiting  Dermatologist  to  the  New  York  City  (Charity)  Hospital ;  Lecturer  on  Genito- 
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Cloth,  $1.25 

Button's  Volumetric  Analysis. 

A  Systematic  Handbook  for  the  Quantitative  Estimation  of  Chemical  Substances  by 
Measure,  Applied  to  Liquids,  Solids,  and  Gases.  Adapted  to  the  Requirements  of 
Pure  Chemical  Research,  Pathological  Chemistry,  Pharmacy,  Metallurgy,  Photog- 
raphy, etc.,  and  for  the  Valuation  of  Substances  Used  in  Commerce,  Agriculture, 
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Swanzy.  Diseases  of  the  Eye  and  their  Treatment. 
A  Handbook  for  Physicians  and  Students.  By  HENRY  R.  SWANZY,  A.M.,  M.B., 
F.R. C.S.I.,  Examiner  in  Ophthalmology,  University  of  Dublin  ;  Surgeon  to  the  National 
Eye  and  Ear  Infirmary  ;  Ophthalmic  Surgeon  to  the  Adelaide  Hospital,  Dublin. 
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"  Is  without  doubt  the  most  satisfactory  manual  we  have  upon  diseases  of  the  eye.  It  occu- 
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encyclopedic  treatises,  which  are  too  extended  and  detailed  to  be  of  special  use  to  the  general 
practitioner." — Chicago  Medical  Recorder.  ' 

Symonds.     Manual  of  Chemistry 

for  Medical  Students.  By  BRANDRETH  SYMONDS,  A.M.,  M.D.,  Assistant  Physician 
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Cloth,  $2.00 

Taft.     Index  of  Dental  Periodical  Literature. 

By  JONATHAN  TAFT,  D.D.S.     8vo.  Cloth,  $2.00 

Tanner's  Memoranda  of  Poisons 

and  their  Antidotes  and  Tests.  By  THOS.  HAWKES  TANNER,  M.D.  Eighth  Edition, 
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MEDICAL   AND   SCIENTIFIC  PUBLICATIONS.  37 


Tavera.     Medicinal  Plants  of  the  Philippines. 

By  T.  H.  PARDO  DE  TAVERA,  Doctor  of  Medicine  in  Faculty  of  Paris  ;  Scientific 
Commissioner  S.M.  in  Philippine  Islands,  etc.  Translated  and  Revised  by  JEROME 
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Taylor.     Practice  of  Medicine. 

By  FREDERICK  TAYLOR,  M.D.,  Physician  to,  and  Lecturer  on  Medicine  at,  Guy's 
Hospital,  London  ;  Physician  to  Evelina  Hospital  for  Sick  Children.  Sixth  Edition, 
Revised.  Cloth,  $4.00 

Taylor  and  Wells.     Diseases  of  Children.     Illustrated. 

A  Manual  for  Students  and  Physicians.  By  JOHN  MADISON  TAYLOR,  A.M.,  M.D., 
Professor  of  Diseases  of  Children,  Philadelphia  Polyclinic  ;  Pediatrist  to  the  Philadel- 
phia Hospital ;  Assistant  Physician  to  the  Children's  Hospital ;  Consulting  Physician 
to  the  Elwyn  and  the  Vineland  Training  Schools  for  Feeble-minded  Children  ; 
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Demonstrator  of  Clinical  Obstetrics,  Jefferson  Medical  College  ;  Chief  Gynecologist, 
Mt.  Sinai  Hospital.  With  Numerous  Illustrations.  Second  Edition,  Revised  and 
Enlarged.  Octavo.  Cloth,  $4.50 

Temperature  Charts 

for  Recording  Temperature,  Respiration,  Pulse,  Day  of  Disease,  Date,  Age,  Sex, 
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Thayer.     Compend  of  General  Pathology. 

Specially  adapted  for  Medical  Students  and  Physicians.  By  A.  E.  THAYER,  M.D., 
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Thorington.     Retinoscopy.     Fourth  Edition. 

(The  Shadow  Test)  in  the  Determination  of  Refraction  at  One  Meter  Distance  with 
the  Plane  Mirror.  By  JAMES  THORINGTON,  A.M.,  M.D.,  Professor  of  Diseases  of  the 
Eye  in  the  Philadelphia  Polyclinic  ;  Ophthalmologist  to  the  Elwyn,  Vineland,  and 
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Anatomy,  Physiology,  and  Care  of  the  Eyes  in  the  Philadelphia  Manual  Training 
Schools,  etc.  51  Illustrations,  several  of  which  are  Colored.  Fourth  Edition,  En- 
larged. i2mo.  Cloth,  #1.00 

Refraction  and  How  to  Refract.     Second  Edition. 

With  200  Illustrations,  most  of  which  are  made  from  Original  Drawings,  and 
13  of  which  are  in  Colors.  Second  Edition,  Revised.  1 2mo.  Cloth,  $1.50 
SYNOPSIS  OF  CONTENTS.— I.  Optics.  II.  The  Eye  ;  The  Standard  Eye ; 
Cardinal  Points  ;  Visual  Angle  ;  Minimum  Visual  Angle  ;  Standard  Acuteness  of 
Vision  ;  Size  of  Retinal  Image,  Accommodation  ;  Mechanism  of  Accommoda- 
tion ;  Far  and  Near  Point  ;  Determination  of  Distant  Vision  and  Near  Point ; 
Amplitude  of  Accommodation  ;  Convergence  ;  Angle  Gamma  ;  Angle  Alpha. 
III.  Ophthalmoscope  ;  Direct  and  Indirect  Method.  IV.  Emmetropia  ;  Hyper- 
opia  ;  Myopia.  V.  Astigmatism  or  Curvature  Ametropia  ;  Tests  for  Astigma- 
tism. VI.  Retinoscopy.  VII.  Muscles.  VIII.  Cycloplegics  ;  Cycloplegia  ; 
Asthenopia ;  Examination  of  the  Eyes.  IX.  How  to  Refract.  X.  Applied 
Refraction.  XI.  Presbyopia ;  Aphakia  ;  Anisometropia ;  Spectacles.  XII. 
Lenses  ;  Spectacle  and  Eye  Glass  Frames  ;  How  to  Take  Measurements  for 
Them  and  How  They  Should  be  Fitted.  Index. 


38  P.   S  LA  KIS  TON'S  SON  &*    CO.1  S 

Thome.     The   Schott   Methods  of  the  Treatment  of  Chronic  Dis- 
eases of  the  Heart. 

With  an  Account  of  the  Nauheim  Baths  and  of  the  Therapeutic  Exercises.  By  W. 
BEZLY  THORNE,  M.D.,  M.R.C.P.  With  Plates  and  Numerous  other  Illustrations. 
Fourth  Edition,  Revised  and  Enlarged.  Octavo.  Just  Ready.  Cloth,  $2.00 

Thresh.     Water  and  Water  Supplies. 

By  JOHN  C.  THRESH,  D.SC.  (Lond.),  M.D.,  D.P.H.  (Cambridge),  Medical  Officer  of 
Health  to  the  Essex  County  Council;  Lecturer  on  Public  Health,  King's  College, 
London  ;  Fellow  of  the  Institute  of  Chemistry  ;  Member  Society  Public  Analysts, 
etc.  Third  Edition,  Revised  and  very  much  Enlarged.  Illustrated.  527  pages. 
I2mo.  Cloth,  $2.00 

Tissier.     Pneumatotherapy  and  Inhalation  Methods. 
See  COHEN,  Physiologic  Therapeutics,  page  10. 

Tomes'  Dental  Anatomy. 

A  Manual  of  Dental  Anatomy,  Human  and  Comparative.  By  C.  S.  TOMES,  D.D.S. 
263  Illustrations.  Fifth  Edition.  i2mo.  Cloth,  $4.00 

Dental  Surgery. 

A  System  of  Dental  Surgery.  By  JOHN  TOMES,  F.R.S.  Fourth  Edition,  Thor- 
oughly Revised  by  C.  S.  TOMES,  D.D.S.  With  289  Illustrations.  I2mo.  717 
pages.  Cloth,  $4.00 

Traube.     Physico-Chemical  Methods. 

By  DR.  J.  TRAUBE,  Privatdocent  in  the  Technical  High  School  of  Berlin.  Author- 
ized Translation  by  W.  D.  HARDIN,  Harrison  Senior  Fellow  in  Chemistry,  University 
of  Pennsylvania.  With  97  Illustrations.  8vo.  Cloth,  $1.50 

Treves.     German-English  Medical  Dictionary. 

By  FREDERICK  TREVES,  F.R.C.S.,  assisted  by  DR.  HUGO  LANG,  B.A.  (Munich). 
I2mo.  Half  Calf,  $3.25 

Physical  Education  :  Its  Effects,  Value,  Methods,  etc.     8vo. 

Cloth,  .75 
Tuke.     Dictionary  of  Psychological  Medicine. 

Giving  the  Definition,  Etymology,  and  Synonyms  of  the  Terms  used  in  Medical  Psy- 
chology, with  the  Symptoms,  Pathology,  and  Treatment  of  the  Recognized  Forms  of 
Mental  Disorders,  together  with  the  Law  of  Lunacy  in  Great  Britain  and  Ireland. 
Edited  by  D.  HACK  TUKE,  M.D.,  LL.D.,  Examiner  in  Mental  Physiology  in  the  Uni- 
versity of  London.  Two  volumes.  Octavo.  Cloth,  $10.00 
"  A  comprehensive,  standard  book." — The  British  Medical  Journal. 

"  It  is  vastly  more  than  a  Dictionary.  It  is  an  elaborate  and  complete  Encyclopaedia  of 
Psychological  Medicine ;  in  fact,  a  small  library  in  itself  on  that  subject.  The  high  expectations 
which  Dr.  Tuke's  work  in  this  field  had  raised  are  more  than  fulfilled.  ...  It  will  be  found 
to  be  a  most  useful  reference  handbook  for  the  alienist  and  student.  The  general  physician  also 
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"We  believe  that  the  student  might  obtain  a  better  knowledge  of  insanity  from  this  work  than 
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Turnbull's  Artificial  Anesthesia. 

A  Manual  of  Anesthetic  Agents  in  the  Treatment  of  Diseases,  also  their  Employment 
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Tests  of  Purity  ;  Treatment  of  Asphyxia  ;  Spasms  of  the  Glottis  ;  Syncope,  etc.  By 
LAURENCE  TURNBULL,  M.D.,  PH.G.,  Aural  Surgeon  to  Jefferson  College  Hospital,  etc. 
Fourth  Edition,  Revised.  54  Illustrations.  I2mo.  Cloth,  $2.50 


MEDICAL   AND   SCIENTIFIC  PUBLICATIONS.  39" 

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Tyson.     The  Practice  of  Medicine.     Second  Edition. 

A  Text-Book  for  Physicians  and  Students,  with  Special  Reference  to  Diagnosis  and 
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*.£*  Sample  Pages  and  Illustrations  sent  free  upon  application. 

Guide  to  the  Examination  of  Urine. 

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Handbook  of  Physical  Diagnosis. 

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Cell  Doctrine. 

Its  History  and  Present  State.     Second  Edition.  Cloth,  $1.50 

United  States  Pharmacopoeia,  1890. 

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40  P.   BLAKISTON'S  SON  &    CO.' S 

, — « 

Ulzer  and  Fraenkel.  Introduction  to  Chemical-Technical  Analysis. 
By  PROF.  F.  ULZER  and  DR.  A.  FRAENKEL,  Directors  of  the  Testing  Laboratory  of 
the  Royal  Technological  Museum,  Vienna.  Authorized  Translation  by  HERMANN 
FLECK,  NAT.SC.D.,  Instructor  in  Chemistry  and  Chemical  Technical  Analysis  in  the 
John  Harrison  Laboratory  of  Chemistry,  University  of  Pennsylvania,  with  an 
Appendix  by  the  Translator  relating  to  Food  Stuffs,  Asphaltum,  and  Paint.  12  Illus- 
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Van-Nliys  on  the  Urine. 

Chemical  Analysis  of  Healthy  and  Diseased  Urine,  Qualitative  and  Quantitative.  By 
T.  C.  VAN  NUYS.  39  Illustrations.  Octavo.  Cloth,  $1.00 

Van  Harlingen  on  Skin  Diseases. 

A  Practical  Manual  of  Diagnosis  and  Treatment,  with  Special  Reference  to  Differential 
Diagnosis.  By  ARTHUR  VAN  HARLINGEN,  M.D.,  Emeritus  Professor  of  Diseases  of 
the  Skin  in  the  Philadelphia  Polyclinic  ;  Dermatologist  to  the  Children's  Hospital. 
Third  Edition,  Revised  and  Enlarged.  With  Formulae  and  Illustrations,  several  being 
in  Colors.  580  pages.  Cloth,  #2.75 

"As  would  naturally  be  expected  from  the  author,  his  views  are  sound,  his  information 

extensive,  and  in  matters  of  practical  detail  the  hand  of  the  experienced  physician  is  everywhere 

visible."  —  The  Medical  News. 

Virchow's  Post-mortem  Examinations. 

A  Description  and  Explanation  of  the  Method  of  Performing  them  in  the  Dead- 
House  of  the  Berlin  Charite  Hospital,  with  Especial  Reference  to  Medico-Legal 
Practice.  By  PROFESSOR  VIRCHOW.  Translated  by  DR.  T.  P.  SMITH.  Illustrated. 
Third  Edition.  Cloth,  .75 

Voswinkel.     Surgical  Nursing. 

A  Manual  for  Nurses.  By  BERTHA  M.  VOSWINKEL,  Graduate  Episcopal  Hospital, 
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Walker.     Students'  Aid  in  Ophthalmology. 

By  GERTRUDE  A.  WALKER,  A.B.,  M.D.,  Clinical  Instructor  in  Diseases  of  the  Eye  at 
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Walsham.     Surgery  :    Its  Theory  and  Practice.     Seventh  Edition. 

For  Students  and  Physicians.  By  WM.  J.  WALSHAM,  M.D.,  F.R.C.S.,  Senior  Assist- 
ant Surgeon  to,  and  Demonstrator  of  Practical  Surgery  in,  St.  Bartholomew's  Hospital ; 
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larged by  100  pages.  With  483  Illustrations  and  28  Skiagrams.  Cloth,  $3.50 

Warren.  Compend  of  Dental  Pathology  and  Dental  Medicine. 
Containing  all  the  most  Noteworthy  Points  of  Interest  to  the  Dental  Student  and  a 
Chapter  on  Emergencies.  By  GEORGE  W.  WARREN,  D.D.S.,  Professor  of  Clinical 
Dentistry  and  Oral  Surgery  ;  Clinical  Chief,  Pennsylvania  College  of  Dental  Surgery, 
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Dental  Prosthesis  and  Metallurgy. 

129  Illustrations.  Cloth,  $1.25 

Weber    and     Hinsdale.       Climatology — Health    Resorts — Mineral 

Springs. 
See  COHEN,  Physiologic  Therapeutics,  page  10. 


MEDICAL  AND   SCIENTIFIC  PUBLICATIONS.  41 


Wells.     Compend  of  Gynecology. 

By  WM.  H.  WELLS,  M.D.,  Demonstrator  of  Clinical  Obstetrics,  Jefferson  Medical 
College,  Philadelphia  ;  Chief  Gynecologist  Mt.  Sinai  Hospital ;  Fellow  of  the  College 
of  Physicians  of  Philadelphia.  Second  Edition,  Revised.  140  Illustrations.  Being 
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Wethered.     Medical  Microscopy. 

A  Guide  to  the  Use  of  the  Microscope  in  Practical  Medicine.  By  FRANK  J.  WETH- 
ERED, M.D.,  M.R.C.P.,  Demonstrator  of  Practical  Medicine,  Middlesex  Hospital  Med- 
ical School ;  Assistant  Physician,  late  Pathologist,  City  of  London  Hospital  for 
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Weyl.     Sanitary  Relations  of  the  Coal-Tar  Colors. 

By  THEODORE  WEYL.  Authorized  Translation  by  HENRY  LEFFMANN,  M.D.,  PH.D 
I2mo.  Cloth,  $1.25 

Whitacre.     Laboratory  Text-Book  of  Pathology. 

By  HORACE  J.  WHITACRE,  M.D.,  Demonstrator  of  Pathology,  Medical  College  of 
Ohio,  Cincinnati.  Illustrated  with  121  Original  Drawings  and  Microphotographs. 
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White.     The  Mouth  and  Teeth.     Illustrated. 

By  J.  W.  WHITE,  M.D.,  D.D.S.  Cloth,  .40 

White  and  Wilcox.     Materia  Medica,  Pharmacy,  Pharmacology,  and 
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A  Handbook  for  Students.  By  W.  HALE  WHITE,  M.D.,  F.R.C.P.,  etc.,  Physician  to, 
and  Lecturer  on  Materia  Medica  and  Therapeutics,  Guy's  Hospital  ;  Examiner  in 
Materia  Medica  to  the  Conjoint  Board,  etc.  Fifth  American  Edition,  Revised  by 
REYNOLD  W.  WILCOX,  M.A.,  M.D.,  LL.D.,  Professor  of  Clinical  Medicine  and  Thera- 
peutics at  the  New  York  Post-Graduate  Medical  School  and  Hospital ;  Visiting  Phy- 
sician, St.  Mark's  Hospital  ;  Assistant  Visiting  Physician,  Bellevue  Hospital.  En- 
larged and  Improved.  I2mo.  Cloth,  $3.00;  Leather,  $3.50 

Williams.     Manual  of  Bacteriology.     Second  Edition. 

By  HERBERT  U.  WILLIAMS,  M.D.,  Professor  of  Pathology  and  Bacteriology,  Medical 
Department,  University  of  Buffalo.  Second  Edition,  Revised  and  Enlarged.  90 
Illustrations.  I2mo.  290  pages.  Cloth,  $1.50 

Wilson.     Handbook  of  Hygiene  and  Sanitary  Science. 

By  GEORGE  WILSON,  M.A.,  M.D.,  F.R.S.E.,  Medical  Officer  of  Health  for  Mid-War- 
wickshire, England.  With  Illustrations.  Eighth  Edition.  I2mo.  Cloth,  $3.00 

Wilson.     The  Summer  and  its  Diseases. 

By  JAMES  C.  WILSON,  M.D.,  Professor  of  the  Practice  of  Medicine  and  Clinical 
Medicine,  Jefferson  Medical  College,  Philadelphia.  Cloth,  .40 

Wilson.     System  of  Human  Anatomy. 

Eleventh  Revised  Edition,  Edited  by  HENRY  EDWARD  CLARK,  M.D.,  M.R.C.S.  492 
Illustrations,  26  Colored  Plates,  and  a  Glossary  of  Terms.  I2mo.  Cloth,  $5.00 

Winckel.     Text-Book  of  Obstetrics. 

Including  the  Pathology  and  Therapeutics  of  the  Puerperal  State.  By  DR.  F. 
WINCKEL,  Professor  of  Gynecology,  Royal  University  Clinic  for  Women  in  Munich. 
Authorized  Translation  by  J.  CLIFTON  EDGAR,  A.M.,  M.D.,  Professor  of  Obstetrics 
and  Clinical  Midwifery,  Cornell  University  Medical  Department,  New  York.  190 
Illustrations.  Octavo.  Cloth,  $5.00  ;  Leather,  $6.00 


42  P.  BLAKISTON'S  SON  6-   CO.' S  PUBLICATIONS. 

Winternitz.     Hydrotherapy — Thermotherapy — Balneology. 
See  COHEN,  Physiologic  Therapeutics,  page  10. 

Wood.     Brain  Work  and  Overwork. 

By  H.  C.  WOOD,  Clinical  Professor  of  Nervous  Diseases,  University  of  Pennsylvania. 
I2mo.  Cloth,  .40 

Woody.     Essentials  of  Medical  and  Clinical  Chemistry. 

With  Laboratory  Exercises.  By  SAMUEL  E.  WOODY,  A.M.,  M.D.,  Professor  of  Chem- 
istry and  Diseases  of  Children  in  the  Medical  Department,  Kentucky  University, 
Louisville.  Fourth  Edition,  Revised  and  Enlarged.  Illustrated.  i2mo.  Cloth,  $1.50 

"  The  fact  that  Prof.  Woody's  little  book  has  reached  a  third  edition  in  such  a  short  time  is 
sufficient  proof  of  its  usefulness  for,  and  demand  by,  the  medical  student.  The  selection  of  the 
material  and  its  plan  of  presentation,  resulting  from  the  author's  large  experience  as  a  practitioner 
and  teacher  of  medical  chemistry,  is  well  intended  to  offer  to  the  student  that  which  is  really  essen- 
tial for  his  limited  college  course,  and,  it  is  to  be  hoped,  a  basis  for  further  instruction  in  the  impor- 
tant branch  of  medical  science." — The  American  Journal  of  Medical  Sciences,  Philadelphia. 

Wright.     Ophthalmology.     New  Edition.      117  Illustrations. 

A  Text-Book  by  JOHN  W.  WRIGHT,  A.M.,  M.D.,  Professor  of  Ophthalmology  and 
Clinical  Ophthalmology  in  Ohio  Medical  University  ;  Ophthalmologist  to  the  Protest- 
ant and  University  Hospitals,  etc.  Second  Edition,  Revised,  Rewritten,  and  Enlarged. 
With  many  new  Illustrations.  Cloth,  $3.00 


1 

MORRIS'  ANATOMY 


THE  STANDARD  TEXT-BOOK 

New  Edition 


Third  Revised  Edition,  Enlarged  and  Improved 

846  Illustrations,  of  which  267  are  Colored 

Octavo.    J328  Pages,    doth,  $6.00;  Leather,  $7.00 

"  Morris'  Anatomy"  was  published  at  a  time  when  methods  of  teaching, 
the  art  of  engraving,  and  distinct  advance  in  anatomical  illustration 
made  desirable  a  new  and  modern  text-book.  The  rapid  sale  of  the  first 
edition,  its  immediate  adoption  as  a  text-book  by  a  large  number  of  medi- 
cal schools,  and  its  purchase  by  physicians  and  surgeons  proved  its  value 
and  made  it  from  the  day  of  publication  a  standard  authority. 


THUMB 
INDEX 
IN  EACH 
COPY 


In  making  this  new  edition  the  editors  and  publishers  have  used  every 
endeavor  to  enhance  its  value.  The  text  has  been  thoroughly  revised  and 
in  many  parts  rewritten ;  the  editor  has  devoted  himself  to  the  task  of 
making  it  a  harmonious  whole;  many  new  illustrations  have  replaced 
those  used  in  the  first  edition,  and  a  large  number  have  been  printed  in 
colors,  while  the  typographical  appearance  has  been  improved  in  several 
particulars. 

The  illustrations,  in  correctness  and  excellence  of  execution,  are  equaled 
by  no  similar  treatise;  about  $1000  having  been  expended  on  new  and 
improved  blocks  for  this  edition  alone. 

"  The  evergrowing  popularity  of  the  book  with  teachers  and  students  is  an  index  of  its  value, 
and  it  may  safely  be  recommended  to  all  interested." — Medical  Record,  New  York. 

"  Of  all  the  text-books  of  moderate  size  on  human  anatomy  in  the  English  language,  Morris 
is  undoubtedly  the  most  up-to-date  and  accurate." — Philadelphia  Medical  Journal. 

V  CIRCULAR  WITH  SAMPLE  PAGES  AND  ILLUSTRATIONS  FREE. 


.From  the  Southern  Clinic. 

"  We  know  of  no  series  of  books  issued  by  any  house  that  so  fully  meets  our  approval  as  these 
?  Quiz-Compends  ?.  They  are  well  arranged,  full,  and  concise,  and  are  really  the  best  line  of  text* 
books  that  could  be  found  for  either  student  or  practitioner." 


BLAKISTON'S  ?QUIZ=COMPENDS? 

The  Best  Series  of  Manuals  for  the  Use  of  Students. 

Price  of  each,  Cloth,  .80.       Interleaved  for  taking  Notes,  81.OO. 


Compends  are  based  on  the  most  popular  text-books  and  the  lectures  of  prominent 
professors,  and  are  kept  constantly  revised,  so  that  they  may  thoroughly  represent  the  present  state 
of  the  subject  upon  which  they  treat.  The  authors  have  had  large  experience  as  Quiz-Masters 
and  attaches  of  colleges,  and  are  well  acquainted  with  the  wants  of  students.  They  are  arranged 
in  the  most  approved  form,  thorough  and  concise,  containing  over  900  illustrations,  inserted 
wherever  they  could  be  used  to  advantage.  Can  be  used  by  students  of  any  college,  and  contain 
information  nowhere  else  collected  in  such  a  condensed  practical  shape. 

No.  i.     HUMAN  ANATOMY.         Sixth  Revised  and  Enlarged   Edition.      Including  Vis- 
'  ce'ral  Anatomy.     Can  be  used  with  either  Morris's  or  Gray's  Anatomy.      117  Illustrations  and 

1  6  Lithographic  Plates  of  Nerves  and  Arteries,  with  Explanatory  Tables,  etc.     By  SAMUEL 

O.  L.  POTTER,  M.D.,  formerly  Professor  of  the  Practice  of  Medicine,  Cooper  Medical  College, 

San  Francisco  ;   Major  and  Brigade  Surgeon,  U.  S.  Vol. 
No.  2.     PRACTICE  OF  MEDICINE.     Part  I.     Sixth  Edition,   Revised,  Enlarged,  and 

Improved.      By  DAN'L   E.   HUGHES,  M.D.,  Physician-in-Chief,  Philadelphia  Hospital;  late 

Demonstrator  of  Clinical  Medicine,  Jefferson  Medical  College,  Philadelphia. 
No.  3.     PRACTICE  OF  MEDICINE.     Part  II.     Sixth  Edition,  Revised,  Enlarged,  and 

Improved.     Same  author  as  No.  2. 
No.  4.     PHYSIOLOGY.     Tenth   Edition,  with  new    Illustrations.     Enlarged   and    Revised. 

By  A.  P.  BRUBAKER,  M.D.,  Professor  of  Physiology  in  the  Pennsylvania  College  of  Dental 

Surgery;  Adjunct  Professor  of  Physiology,  Jefferson  Medical  College,  Philadelphia. 
No.  5.     OBSTETRICS.      Seventh   Edition.      By  HENRY  G.   LANDIS,   M.D.      Revised  and 

Edited  by  WM.    H.   WELLS,   M.D.,   Demonstrator  of   Clinical  Obstetrics,  Jefferson  Medical 

College,  Philadelphia.      Enlarged.      52  Illustrations. 
No.  6.     MATERIA     M  E  D  I  C  A,    THERAPEUTICS,    AND    PRESCRIPTION 

WRITING.      Sixth  Revised  Edition.     Same  author  as  No.  I. 
No.    7.     GYNECOLOGY.     Second  Edition.      By  WM.    H.    WELLS,   M.D.,   Demonstrator  of 

Clinical  Obstetrics,  Jefferson  Medical  College,  Philadelphia.      140  Illustrations. 

No.  8.  DISEASES  OF  THE  EYE  AND  REFRACTION.  Second  Edition.  Includ- 
ing Treatment  and  Surgery  and  a  Section  on  Local  Therapeutics.  By  GEORGE  M.  GOULD, 
M.D.,  Editor  Philadelphia  Medical  Journal,  and  WT.  L.  PYLE,  M.D.  ,  Assistant  Surgeon,  Wills 
Eye  Hospital.  With  Formulae,  Glossary,  several  useful  Tables,  and  109  Illustrations. 

No.  g.  SURGERY,  Minor  Surgery,  and  Bandaging.  Fifth  Edition,  Enlarged  and  Im- 
proved. By  ORVILLE  HORWITZ,  B.S.,  M.D.,  Clinical  Professor  of  Genito-  Urinary  Surgery 
and  Venereal  Diseases  in  Jefferson  Medical  College  ;  Surgeon  to  Philadelphia  Hospital,  etc. 
With  98  Formulae  and  167  Illustrations. 

No.  10.  MEDICAL  CHEMISTRY.  Fourth  Edition.  Including  Urinalysis,  Chemistry  of 
Milk,  Blood,  etc.  By  HENRY  LEFFMANN,  M.D.,  Professor  of  Chemistry  in  Pennsylvania 
College  of  Dental  Surgery  and  in  the  Woman's  Medical  College,  Philadelphia. 

No.  ii.  PHARMACY.  Fifth  Edition.  Based  upon  Professor  Remington's  Text-Book  of 
Pharmacy.  By  F.  E.  STEWART,  M.D.,  PH.G.,  late  Quiz-Master  in  Pharmacy  and  Chemistry, 
Philadelphia  College  of  Pharmacy  ;  Lecturer  at  Jefferson  Medical  College. 

No.  12.  VETERINARY  ANATOMY  AND  PHYSIOLOGY.  Illustrated.  By  WM. 
R.  BALLOU,  M.D.,  Professor  of  Equine  Anatomy  at  New  York  College  of  Veterinary  Sur- 
geons ;  Physician  to  Bellevue  Dispensary,  etc.  With  29  graphic  Illustrations. 

No.  13.  DENTAL  PATHOLOGY  AND  DENTAL  MEDICINE.  Third  Edition, 
Illustrated.  By  GEORGE  W.  WARREN,  D.D.S.,  Pennsylvania  College  of  Dental  Surgery. 

No.  14.  DISEASES  OF  CHILDREN.  Colored  Plate.  By  MARCUS  P.  HATFIELD, 
Professor  of  Diseases  of  Children,  Chicago  Medical  College.  Second  Edition,  Preparing. 

No.  15.     GENERAL  PATHOLOGY.  Illustrated.    By  A.  E.  THAYER,  M.D.,  etc.  Just  Ready. 

No.  16.  DISEASES  OF  THE  SKIN.  By  JAY  F.  SCHAMBERG,  M.D,  Professor  of  Skin 
Diseases,  Philadelphia  Polyclinic.  Second  Edition,  Revised.  105  Illustrations. 

No.  17.     HISTOLOGY.     Illustrated.     By  H.  H.  GUSHING,  M.D.  Preparing. 

No.  18.     SPECIAL  PATHOLOGY.     Illustrated.     By  same  author  as  No.  15.     Just  Ready. 

43 


PRACTICAL  GYNECOLOGY 

A  Modern  Comprehensive  Text-Book 
By  E.  E.  MONTGOMERY,  M.D. 

Professor  of  Gynecology,  Jefferson  Medical  College?   Gynecologist  to  the  Jefferson  Medical 

College  and  St.  Joseph's  Hospitals;   Consulting  Gynecologist  to 

the  Philadelphia  Lying-in  Charity 

WITH  FIVE  HUNDRED  AND  TWENTY-SEVEN 
ILLUSTRATIONS 

Nearly  all  of  which  have  been  Drawn  and  Engraved  Specially  for  this 
Work,  for  the  most  part  from  Original  Sources 

OCTAVO.    819  PAGES.    CLOTH,  $5.00 ;  LEATHER,  $6.00 


From  THE  JOURNAL  OF  THE  AMERICAN  MEDICAL  ASSOCIATION. 

"  Fashion  in  medical  book-making  seems  to  be  running  to  the  composite,  which 
may  be  advantageous  and  the  means  of  producing  a  better  book  than  one  written  by 
an  individual.  It  may  be  the  old-fashioned  notions  of  the  reviewer,  but  he  belives  in 
the  old  idea  of  one  book,  one  author,  and  he  should  have  all  the  responsibility,  all  the 
criticism,  and  all  the  glory  that  attach  to  it.  The  composite  is  likely  to  be  written 
under  a  '  rush '  order — so  much  space,  in  so  much  time,  for  so  much  money.  The  work 
before  tis  is  the  work  of  one  individual,  and  the  personality  of  that  individual  is  evident 
through  the  whole  book.  ...  The  result  shows  painstaking  effort  in  every  detail, 
in  conciseness  of  statements,  in  arrangement  of  subjects,  and  in  the  systematic  order 
and  completeness  in. which  each  is  considered.  .  .  .  The  author  is  neither  too 
radical  nor  too  conservative  in  his  consideration  of  the  conditions  that  may  need  radical 
operations.  In  the  introduction  he  tells  us  that  the  true  gynecologist  must  be  '  so  con 
servative  that  he  will  sacrifice  no  organ  whose  physiologic  integrity  is  capable  of  being 
restored ;  so  bold  and  courageous  that  his  patient  shall  not  forfeit  her  opportunity  for 
life  or  restored  health  through  his  failure  to  assume  the  responsibility  of  any  operative 
procedure  necessary  to  secure  the  object.'  This  is  the  basal  idea  that  permeates  the 
book  :  the  ultra-radical  operator  will  find  no  endorsement,  and  the  'tinkering '  gynecologist— 
he  who  treats  all  diseases  of  women  by  means  of  a  pledget  of  cotton  and  a  speculum — 
no  encouragement  in  its  pages* 

"The  book  is  one  that  can  be  recommended  to  the  student,  to  the  general  practi- 
tioner— who  must  sometimes  be  a  gynecologist  to  a  certain  extent  whether  he  will  or  not 
— and  to  the  specialist,  as  an  ideal  and  in  every  way  complete  work  on  the  gynecology  of 
to-day — a  practical  work  for  practical  workers." 


DESCRIPTIVE  CIRCULAR  UPON  APPLICATION. 

44 


JACOBSON'S 

OPERATIONS   OF   SURGERY 

The  Operations  of  Surgery.  By  W.  H.  A.  JACOBSON, 
F.R.C.S.,  Surgeon  to  Guy's  Hospital,  Consulting  Surgeon 
Royal  Hospital  for  Children  and  Women,  Member  Court 
of  Examiners  Royal  College  of  Surgeons,  Joint  Editor 
Annals  of  Surgery;  and  F.  J.  STEWARD,  F.R.C.S.,  Assistant 
Surgeon  Guy's  Hospital  and  to  the  Hospital  for  Sick 
Children.  Fourth  Edition,  Revised,  Enlarged  and  Im- 
proved. 550  Illustrations;  Two  Volumes ;  Octavo;  1524 
Pages.  Cloth,  $10.00;  Sheep  or  Half  Morocco,  $12.00 

This  printing  has  been  increased  in  size  over  the  former  edition 
by  200  pages  and  contains  150  additional  illustrations. 

PRESS  NOTICES    OF  FORMER   EDITIONS 

' '  Far  more  than  a  mere  guide  to  operating,  it  is  essentially  a  clinical  work  and  in 
that  lies  one  of  its  conspicuous  merits."  —  The  London  Lancet. 

"The  author  proves  himself  a  judicious  operator  as  shown  by  his  choice  of 
methods,  and  by  the  emphasis  with  which  he  refers  to  the  different  dangers  and  com- 
plications which  may  arise  to  mar  success  or  jeopardize  life." — New  York  Medical 
Record. 

"  Many  of  the  difficulties  met  with  at  the  time  of  an  operation,  and  the  troubles 
ensuing  after  an  operation,  which  are  only  known  to  the  practical  surgeon,  are  brought 
out  prominently  in  this  book." — Boston  Medical  and  Surgical  Journal. 

"The  important  anatomical  points  are  clearly  set  forth,  the  conditions  indicating  or 
contraindicating  operative  interference  are  given,  the  details  ot  the  operations  them- 
selves are  brought  forward  prominently,  and  frequently  the  after-treatment  is  considered. 
Herein  is  one  of  the  strong  points  of  the  book." — New  York  Medical  Journal. 

:%.:::  Jacobson's  Operations  of  Surgery  is  not  intended  only  for 
those  of  great  surgical  experience  or  skill,  but  is  intended  largely  as 
an  authoritative  guide  for  the  GENERAL  PHYSICIAN  and  HOSPITAL 
RESIDENT  who,  in  emergencies  where  immediate  surgical  intervention 
is  demanded,  must  act  quickly,  and  often  rely  solely  upon  his  own 
judgment. 

Camprehensive  in  scope,  exhaustive  in  detail,  rich  in  its  exposi- 
tion of  the  latest  and  most  uniformly  successful  methods  in  operating, 
and  modern  throughout  in  its  treatment  of  each  branch  of  surgical 
work,  particularly  that  of  abdominal  surgery,  this  book  easily  ranks 
among  the  very  foremost  works  in  its  particular  field. 

45 


CARPENTER  ON  THE  MICROSCOPE 


AND  ITS  REVELATIONS 


EIGHTH 


Edited  by  W.  H.  Dallin^cr,  D.Sc.,  D.C.L,   F.R.S. 


With  23  Plates  and  nearly  900  Engravings 


OCTAVO.     1181  PAGES.     CLOTH,  $8.00;   HALF  MOROCCO,  $9.00 


***  Eight  of  the  chapters  have  been  entirely  rewritten  and  the  text 
throughout  reconstructed,  enlarged,  and  revised  with  the  aid  and  advice 
of  E.  M*  Nelson,  ex-President  of  The  Royal  Microscopical  Society; 
Arthur  Bolles  Lee,  author  of  "The  Microtomist's  Vade  Mecum";  Dr*  E. 
Crookshank,  the  well-known  Bacteriologist;  Prof*  T.  Bonney,  F*R*S.; 
W.  J*  Pope,  F*I*C*,  F*C.S«,  etc*,  Chemist  to  the  Goldsmith's  Technical 
Institute;  Prof*  A.  W.  Bennett,  Lecturer  on  Botany  at  St*  Thomas'  Hos- 
pital ;  and  F*  Jeffrey  Bell,  Professor  of  Comparative  Anatomy  and  Zoology, 
King's  College,  London. 

*^.*A  thorough  and  complete  revision  of  the  entire  text  has 
been  made;  eight  chapters  have  been  entirely  reconstructed,  and 
everything  of  importance  to  Microscopy  which  has  transpired  in 
the  interval  has  been  noted.  This  applies  to  the  theory  of  the 
Microscope  as  well  as  to  its  use*  Many  new  illustrations  have 
been  included  and  it  has  been  very  materially  increased  in  size. 


••  CARPENTER "  is  the  only  complete  and  exhaustive  modern  work  on 

the  Science  of  Microscopy 

46 


Diseases  of  tke  Digestive  Tract 

Their  Special  Pathology,  Diagnosis,  and  Treatment*  With 
Sections  on  Anatomy  and  Physiology,  Analysis  of  Stomach 
and  Intestinal  Contents,  Secretions,  Feces,  Urine,  Bacteria, 
Parasites,  etc*,  Surgery,  Dietetics,  Diseases  of  the  Rectum,  etc* 

AN   EXHAUSTIVE   SYSTEMATIC   TREATISE 

By  JOHN  C.  HEMMETER,  M.D. 

Professor  in  the  Medical  Department  of  the  University  of  Maryland  j  Consultant  to  the  University  Hospital  and 

Director  of  the  Clinical  Laboratory,  etc.;  formerly  Clinical  Professor  of  Medicine 

at  the  Baltimore  Medical  College,  etc. 


DISEASES   OF  THE  STOMACH.    Third  Edition. 

With   15   Plates   and  41    other  Illustrations,  some  of  which 
are  printed  in  Colors.      Octavo.      894  pages. 

Cloth,  $6.00  ;  Sheep,  $7.00 

DISEASES   OF  THE  INTESTINES.    Two  Volumes. 

With   19   Plates  and   no  other  Illustrations,  some  of  which 
are  printed  in  Colors.      Octavo.      1421  pages. 

VOL.  I.  Anatomy,  Physiology,  Pathology,  Diagnosis,  Thera- 
peutics, Intestinal  Clinic,  etc.  Cloth,  $5.00;  Sheep,  $6.00 

VOL.  II.  Appendicitis,  Occlusions,  Intestinal  Surgery,  En- 
teroptosis,  Infectious  Granulomata,  Neuroses,  Parasites,  Dis- 
eases of  the  Rectum,  etc.  Cloth,  $5.00 ;  Sheep,  $6.00 

*^*  These  books  form  a  complete  treatise  on  Diseases  of  the  Digestive  Tract. 
The  subject  is  covered  thoroughly  and  systematically  by  an  author  of  well-known 
reputation  and  ability.  The  results  of  recent  investigation,  by  which  so  much 
progress  has  been  made  in  the  Pathology,  Diagnosis,  and  Medical  and  Surgical 
Treatment  of  disorders  of  the  intestinal  tract,  make  their  issue  at  this  time  of 
special  importance.  They  are  handsomely  illustrated,  exhaustive,  and  written 
for  the  general  practitioner,  taking  into  special  consideration  American  habits  of 
living,  diet,  and  climate. 

"  W*  wish  to  express  unqualified  approval  of  *he  tendency  which  is  shown  to  emphasize  the 
simple  and  more  practical  methods  of  diagnosis." — New  York  Medical  Journal,  Review  of  "  Dis- 
eases of  the  Stomach." 


DESCRIPTIVE  CIRCULAR  UPON  APPLICATION 

47 


IN    PRESS 


EDGAR'S  OBSTETRICS 


A  NEW  TEXT-BOOK 


By  J.  CLIFTON  EDGAR,  M.D. 

Professor  of  Obstetrics,  Medical  Department  of  Cornell  University,  New  York  City?  Physician  to  Mothers'  and 
Babies'  Hospital  and  to  the  Emergency  Hospital,  etc. 


Octavo,  about  JOOO  Pages;  900  Illustrations 


THE  ILLUSTRATIONS  in  Edgar's  Obstetrics  surpass  in  number,  in  artistic 
beauty  and  in  practical  worth  those  in  any  book  of  similar  character.  They  are 
largely  from  original  sources.  Those  which  follow  other  works  have  been 
redrawn  with  modifications  so  that  the  entire  series  is  new.  All  have  been  drawn 
by  artists  of  long  experience  in  this  department  of  medical  illustration,  and 
whenever  of  advantage  to  do  so  are  reproduced  at  a  stated  scale. 

No  attempt  has  been  made  at  display.  When  a  small  cut  serves  every  pur- 
pose drawings  are  not  reproduced  to  occupy  a  large  space;  when  black  and  white 
are  equally  expressive  an  elaborate  colored  plate  has  not  been  used.  So  far 
as  possible,  cuts  have  been  inserted  in  the  text  where  they  are  wanted  and  where 
the  eye  catches  them  at  the  place  the  text  explains  them.  Relative  importance 
has  determined  the  selection,  the  size,  and  the  character  of  each  figure.  There 
are  many  explanatory  diagrams  which  add  greatly  to  the  teaching  values  of  the 
pictures.  The  aim  of  author,  artist,  and  publisher  has  been  to  make  a  series  of 
pictures  useful  to  the  student  and  reader,  and  no  time,  labor,  or  money  has 
been  spared  to  gain  this  end.  The  lack  of  uniformity  in  quality  and  failure  to 
observe  scale — the  great  faults  in  books  on  this  subject — have  been  kept  constantly 
in  mind,  and  every  endeavor  has  been  made  to  avoid  similar  defects. 

THE  TEXT  has  been  prepared  with  great  care.  The  author's  extensive 
experience  in  hospital  and  private  practice  and  as  a  teacher,  his  cosmopolitan 
knowledge  of  literature  and  methods,  and  an  excellent  judgment  based  upon  all 
these  fit  him  specially  to  prepare  what  must  be  a  standard  work  for  both  students 
and  physicians. 

In  the  text  as  in  the  illustrating,  uniformity  and  consistency  have  been  kept 
constantly  in  view.  The  subjects  of  monstrosities  and  malformations,  for  example, 
do  not  take  up  space  which  could  be  better  used  for  more  practical  and  useful 
matters,  though  these  topics  like  others  of  their  class  receive  due  consideration 
and  are-  illustrated  by  a  very  complete  series  of  small  figures.  Nothing  of 
importance  remains  unsaid,  and  the  relative  value  of  each  subject  has  been  care- 
fully planned  out  and  fixed  by  deliberate  thought.  The  author's  reputation  is 
sufficient  guarantee  of  the  merit  of  this  book ;  the  publishers,  however,  ask  a 
comparison  with  other  works,  with  confidence  that  this  will  be  found  the  most 
useful. 

48 


NOV.  8  1902 


14  DAY  USE 


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